Combinations of Pyrazole Kinase Inhibitors

ABSTRACT

The invention provides a combination comprising a cytotoxic compound, a signalling inhibitor, an ancillary agent, or two or more further anti-cancer agents, and a compound having the formula (Ib): 
     
       
         
         
             
             
         
       
     
     or salts or tautomers or N-oxides or solvates thereof;
 
wherein
         X is a group R 1 -A-NR 4 —;   A is a bond, C═O, NR g (C═O) or O(C═O) wherein R g  is hydrogen or C 1-4  hydrocarbyl optionally substituted by hydroxy or C 1-4  alkoxy;   Y is a bond or an alkylene chain of 1, 2 or 3 carbon atoms in length;   R 1  is a carbocyclic or heterocyclic group having from 3 to 12 ring members; or a C 1-8  hydrocarbyl group optionally substituted by one or more substituents selected from fluorine, hydroxy, C 1-4  hydrocarbyloxy, amino, mono- or di-C 1-4  hydrocarbylamino, and carbocyclic or heterocyclic groups having from 3 to 12 ring members, and wherein 1 or 2 of the carbon atoms of the hydrocarbyl group may optionally be replaced by an atom or group selected from O, S, NH, SO, SO 2 ;   R 2  is hydrogen; halogen; C 1-4  alkoxy (e.g. methoxy); or a C 1-4  hydrocarbyl group optionally substituted by halogen (e.g. fluorine), hydroxyl or C 1-4  alkoxy (e.g. methoxy);   R 3  is selected from carbocyclic and heterocyclic groups having from 3 to 12 ring members; and   R 4  is hydrogen or a C 1-4  hydrocarbyl group optionally substituted by halogen (e.g. fluorine), hydroxyl or C 1-4  alkoxy (e.g. methoxy).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. Nos.11/814,456, 11/814,461, 11/814,455, and 12/373,713, the entire contentsof which are incorporated herein by reference.

U.S. application Ser. No. 11/814,456 (which published on Jul. 3, 2008 asUS 2008-0161251 A1), was filed Jul. 20, 2007 as a national phase filingunder 35 USC §371 of PCT International Application PCT/GB2006/000204,filed Jan. 20, 2006 and published under PCT Article 21(2) in English asWO 2006/077424 on Jul. 27, 2006. PCT/GB2006/000204 claimed priority fromU.S. provisional applications 60/645,987; 60/645,986; 60/646,113;60/645,976; and 60/645,975; all filed Jan. 21, 2005. The entire contentsof each of the prior applications are incorporated herein by reference.

U.S. application Ser. No. 11/814,461 (which published on Feb. 5, 2009 asUS 2009-0036435 A1) was filed Jul. 20, 2007 as a national phase filingunder 35 USC §371 of PCT International Application PCT/GB2006/000210,filed Jan. 20, 2006, and published under PCT Article 21(2) in English asWO 2006/077428 on Jul. 27, 2006. PCT/GB2006/000210 claimed priority fromU.S. provisional applications 60/645,988; 60/645,974; 60/646,216;60/646,001; and 60/646,003; all filed Jan. 21, 2005. The entire contentsof each of the prior applications are incorporated herein by reference.

U.S. application Ser. No. 11/814,455 (which published on July 3, 2008 asUS 2008-0161355 A1) was filed Jul. 20, 2007 as a national phase filingunder 35 USC §371 of PCT International Application PCT/GB2006/000206,filed Jan. 20, 2006, and published under PCT Article 21(2) in English asWO 2006/077425 on Jul. 27, 2006. PCT/GB2006/000206 claimed priority fromU.S. provisional application 60/645,963, filed Jan. 21, 2005. The entirecontents of each of the prior applications are incorporated herein byreference.

U.S. application Ser. No. 12/373,713 (which published on Oct. 22, 2009as US 2009-0263398 A1) was filed Jan. 13, 2009 as a national phasefiling under 35 U.S.C. §371 of PCT International ApplicationPCT/GB2007/002640, filed Jul. 13, 2007, and published under PCT Article21(2) in English as WO 2008/007113 on Jan. 17, 2008. PCT/GB2007/002640claimed priority from U.S. provisional patent application No. 60/831,043filed on Jul. 14, 2006. The entire contents of each of the priorapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The compounds of Formula (I) and subgroups thereof and the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide and the hydrochloric acid addition salt thereof aredisclosed in our earlier International patent application numberPCT/GB2004/003179 (Publication No. WO 2005/012256) as being inhibitorsof Cyclin Dependent Kinases (CDK kinases) and Glycogen Synthase Kinase-3(GSK3).

The methanesulphonic acid and acetic acid addition salts of compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide and crystals thereof and method of making them aredisclosed in our earlier applications U.S. Ser. No. 60/645,973 and GB0501475.8.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a wide variety of signaltransduction processes within the cell (Hardie, G. and Hanks, S. (1995)The Protein Kinase Facts Book. I and II, Academic Press, San Diego,Calif.). The kinases may be categorized into families by the substratesthey phosphorylate (e.g., protein-tyrosine, protein-serine/threonine,lipids, etc.). Sequence motifs have been identified that generallycorrespond to each of these kinase families (e.g., Hanks, S. K., Hunter,T., FASEB J., 9:576-596 (1995); Knighton, et al., Science, 253:407-414(1991); Hiles, et al., Cell, 70:419-429 (1992); Kunz, et al., Cell,73:585-596 (1993); Garcia-Bustos, et al., EMBO J., 13:2352-2361 (1994)).

Protein kinases may be characterized by their regulation mechanisms.These mechanisms include, for example, autophosphorylation,transphosphorylation by other kinases, protein-protein interactions,protein-lipid interactions, and protein-polynucleotide interactions. Anindividual protein kinase may be regulated by more than one mechanism.

Kinases regulate many different cell processes including, but notlimited to, proliferation, differentiation, apoptosis, motility,transcription, translation and other signalling processes, by addingphosphate groups to target proteins. These phosphorylation events act asmolecular on/off switches that can modulate or regulate the targetprotein biological function. Phosphorylation of target proteins occursin response to a variety of extracellular signals (hormones,neurotransmitters, growth and differentiation factors, etc.), cell cycleevents, environmental or nutritional stresses, etc. The appropriateprotein kinase functions in signalling pathways to activate orinactivate (either directly or indirectly), for example, a metabolicenzyme, regulatory protein, receptor, cytoskeletal protein, ion channelor pump, or transcription factor. Uncontrolled signalling due todefective control of protein phosphorylation has been implicated in anumber of diseases, including, for example, inflammation, cancer,allergy/asthma, disease and conditions of the immune system, disease andconditions of the central nervous system, and angiogenesis.

Cyclin Dependent Kinases

The process of eukaryotic cell division may be broadly divided into aseries of sequential phases termed G1, S, G2 and M. Correct progressionthrough the various phases of the cell cycle has been shown to becritically dependent upon the spatial and temporal regulation of afamily of proteins known as cyclin dependent kinases (cdks) and adiverse set of their cognate protein partners termed cyclins. Cdks arecdc2 (also known as cdk1) homologous serine-threonine kinase proteinsthat are able to utilise ATP as a substrate in the phosphorylation ofdiverse polypeptides in a sequence dependent context. Cyclins are afamily of proteins characterised by a homology region, containingapproximately 100 amino acids, termed the “cyclin box” which is used inbinding to, and defining selectivity for, specific cdk partner proteins.

Modulation of the expression levels, degradation rates, and activationlevels of various cdks and cyclins throughout the cell cycle leads tothe cyclical formation of a series of cdk/cyclin complexes, in which thecdks are enzymatically active. The formation of these complexes controlspassage through discrete cell cycle checkpoints and thereby enables theprocess of cell division to continue. Failure to satisfy thepre-requisite biochemical criteria at a given cell cycle checkpoint,i.e. failure to form a required cdk/cyclin complex, can lead to cellcycle arrest and/or cellular apoptosis. Aberrant cellular proliferation,as manifested in cancer, can often be attributed to loss of correct cellcycle control. Inhibition of cdk enzymatic activity therefore provides ameans by which abnormally dividing cells can have their divisionarrested and/or be killed. The diversity of cdks, and cdk complexes, andtheir critical roles in mediating the cell cycle, provides a broadspectrum of potential therapeutic targets selected on the basis of adefined biochemical rationale.

Progression from the G1 phase to the S phase of the cell cycle isprimarily regulated by cdk2, cdk3, cdk4 and cdk6 via association withmembers of the D and E type cyclins. The D-type cyclins appearinstrumental in enabling passage beyond the G1 restriction point, whereas the cdk2/cyclin E complex is key to the transition from the G1 to Sphase. Subsequent progression through S phase and entry into G2 isthought to require the cdk2/cyclin A complex. Both mitosis, and the G2to M phase transition which triggers it, are regulated by complexes ofcdk1 and the A and B type cyclins.

During G1 phase Retinoblastoma protein (Rb), and related pocket proteinssuch as p130, are substrates for cdk(2, 4, & 6)/cyclin complexes.Progression through G1 is in part facilitated by hyperphosphorylation,and thus inactivation, of Rb and p130 by the cdk(4/6)/cyclin-Dcomplexes. Hyperphosphorylation of Rb and p130 causes the release oftranscription factors, such as E2F, and thus the expression of genesnecessary for progression through G1 and for entry into S-phase, such asthe gene for cyclin E. Expression of cyclin E facilitates formation ofthe cdk2/cyclin E complex which amplifies, or maintains, E2F levels viafurther phosphorylation of Rb. The cdk2/cyclin E complex alsophosphorylates other proteins necessary for DNA replication, such asNPAT, which has been implicated in histone biosynthesis. G1 progressionand the G1/S transition are also regulated via the mitogen stimulatedMyc pathway, which feeds into the cdk2/cyclin E pathway. Cdk2 is alsoconnected to the p53 mediated DNA damage response pathway via p53regulation of p21 levels. p21 is a protein inhibitor of cdk2/cyclin Eand is thus capable of blocking, or delaying, the G1/S transition. Thecdk2/cyclin E complex may thus represent a point at which biochemicalstimuli from the Rb, Myc and p53 pathways are to some degree integrated.Cdk2 and/or the cdk2/cyclin E complex therefore represent good targetsfor therapeutics designed at arresting, or recovering control of, thecell cycle in aberrantly dividing cells.

The exact role of cdk3 in the cell cycle is not clear. As yet no cognatecyclin partner has been identified, but a dominant negative form of cdk3delayed cells in G1, thereby suggesting that cdk3 has a role inregulating the G1/S transition.

Although most cdks have been implicated in regulation of the cell cyclethere is evidence that certain members of the cdk family are involved inother biochemical processes. This is exemplified by cdk5 which isnecessary for correct neuronal development and which has also beenimplicated in the phosphorylation of several neuronal proteins such asTau, NUDE-1, synapsin1, DARPP32 and the Munc18/Syntaxin1A complex.Neuronal cdk5 is conventionally activated by binding to the p35/p39proteins. Cdk5 activity can, however, be deregulated by the binding ofp25, a truncated version of p35. Conversion of p35 to p25, andsubsequent deregulation of cdk5 activity, can be induced by ischemia,excitotoxicity, and β-amyloid peptide. Consequently p25 has beenimplicated in the pathogenesis of neurodegenerative diseases, such asAlzheimer's, and is therefore of interest as a target for therapeuticsdirected against these diseases.

Cdk7 is a nuclear protein that has cdc2 CAK activity and binds to cyclinH. Cdk7 has been identified as component of the TFIIH transcriptionalcomplex which has RNA polymerase II C-terminal domain (CTD) activity.This has been associated with the regulation of HIV-1 transcription viaa Tat-mediated biochemical pathway. Cdk8 binds cyclin C and has beenimplicated in the phosphorylation of the CTD of RNA polymerase II.Similarly the cdk9/cyclin-T1 complex (P-TEFb complex) has beenimplicated in elongation control of RNA polymerase II. PTEF-b is alsorequired for activation of transcription of the HIV-1 genome by theviral transactivator Tat through its interaction with cyclin T1. Cdk7,cdk8, cdk9 and the P-TEFb complex are therefore potential targets foranti-viral therapeutics.

At a molecular level mediation of cdk/cyclin complex activity requires aseries of stimulatory and inhibitory phosphorylation, ordephosphorylation, events. Cdk phosphorylation is performed by a groupof cdk activating kinases (CAKs) and/or kinases such as wee1, Myt1 andMik1. Dephosphorylation is performed by phosphatases such as cdc25(a &c), pp2a, or KAP.

Cdk/cyclin complex activity may be further regulated by two families ofendogenous cellular proteinaceous inhibitors: the Kip/Cip family, or theINK family. The INK proteins specifically bind cdk4 and cdk6. p16^(ink4)(also known as MTS1) is a potential tumour suppressor gene that ismutated, or deleted, in a large number of primary cancers. The Kip/Cipfamily contains proteins such as p21^(Cip1), p27^(Kip1) and p57^(kip2).As discussed previously p21 is induced by p53 and is able to inactivatethe cdk2/cyclin(E/A) and cdk4/cyclin(D1/D2/D3) complexes. Atypically lowlevels of p27 expression have been observed in breast, colon andprostate cancers. Conversely over expression of cyclin E in solidtumours has been shown to correlate with poor patient prognosis. Overexpression of cyclin D1 has been associated with oesophageal, breast,squamous, and non-small cell lung carcinomas.

The pivotal roles of cdks, and their associated proteins, inco-ordinating and driving the cell cycle in proliferating cells havebeen outlined above. Some of the biochemical pathways in which cdks playa key role have also been described. The development of monotherapiesfor the treatment of proliferative disorders, such as cancers, usingtherapeutics targeted generically at cdks, or at specific cdks, istherefore potentially highly desirable. Cdk inhibitors could conceivablyalso be used to treat other conditions such as viral infections,autoimmune diseases and neuro-degenerative diseases, amongst others. Cdktargeted therapeutics may also provide clinical benefits in thetreatment of the previously described diseases when used in combinationtherapy with either existing, or new, therapeutic agents. Cdk targetedanticancer therapies could potentially have advantages over many currentantitumour agents as they would not directly interact with DNA andshould therefore reduce the risk of secondary tumour development.

Glycogen Synthase Kinase

Glycogen Synthase Kinase-3 (GSK3) is a serine-threonine kinase thatoccurs as two ubiquitously expressed isoforms in humans (GSK3α & betaGSK3β). GSK3 has been implicated as having roles in embryonicdevelopment, protein synthesis, cell proliferation, celldifferentiation, microtubule dynamics, cell motility and cellularapoptosis. As such GSK3 has been implicated in the progression ofdisease states such as diabetes, cancer, Alzheimer's disease, stroke,epilepsy, motor neuron disease and/or head trauma. Phylogenetically GSK3is most closely related to the cyclin dependent kinases (CDKs).

The consensus peptide substrate sequence recognised by GSK3 is(Ser/Thr)-X-X-X-(pSer/pThr), where X is any amino acid (at positions(n+1), (n+2), (n+3)) and pSer and pThr are phospho-serine andphospho-threonine respectively (n+4). GSK3 phosphorylates the firstserine, or threonine, at position (n). Phospho-serine, orphospho-threonine, at the (n+4) position appears necessary for primingGSK3 to give maximal substrate turnover. Phosphorylation of GSK3α atSer21, or GSK3β at Ser9, leads to inhibition of GSK3. Mutagenesis andpeptide competition studies have led to the model that thephosphorylated N-terminus of GSK3 is able to compete withphospho-peptide substrate (S/TXXXpS/pT) via an autoinhibitory mechanism.There are also data suggesting that GSK3α and GSKβ may be subtlyregulated by phosphorylation of tyrosines 279 and 216 respectively.Mutation of these residues to a Phe caused a reduction in in vivo kinaseactivity. The X-ray crystallographic structure of GSK3β has helped toshed light on all aspects of GSK3 activation and regulation.

GSK3 forms part of the mammalian insulin response pathway and is able tophosphorylate, and thereby inactivate, glycogen synthase. Upregulationof glycogen synthase activity, and thereby glycogen synthesis, throughinhibition of GSK3, has thus been considered a potential means ofcombating type II, or non-insulin-dependent diabetes mellitus (NIDDM): acondition in which body tissues become resistant to insulin stimulation.The cellular insulin response in liver, adipose, or muscle tissues istriggered by insulin binding to an extracellular insulin receptor. Thiscauses the phosphorylation, and subsequent recruitment to the plasmamembrane, of the insulin receptor substrate (IRS) proteins. Furtherphosphorylation of the IRS proteins initiates recruitment ofphosphoinositide-3 kinase (PI3K) to the plasma membrane where it is ableto liberate the second messenger phosphatidylinosityl3,4,5-trisphosphate (PIP3). This facilitates co-localisation of3-phosphoinositide-dedependent protein kinase 1 (PDK1) and proteinkinase B (PKB or Akt) to the membrane, where PDK1 activates PKB. PKB isable to phosphorylate, and thereby inhibit, GSK3α and/or GSKβ throughphosphorylation of Ser9, or ser21, respectively. The inhibition of GSK3then triggers upregulation of glycogen synthase activity. Therapeuticagents able to inhibit GSK3 may thus be able to induce cellularresponses akin to those seen on insulin stimulation. A further in vivosubstrate of GSK3 is the eukaryotic protein synthesis initiation factor2B (eIF2B). eIF2B is inactivated via phosphorylation and is thus able tosuppress protein biosynthesis. Inhibition of GSK3, e.g. by inactivationof the “mammalian target of rapamycin” protein (mTOR), can thusupregulate protein biosynthesis. Finally there is some evidence forregulation of GSK3 activity via the mitogen activated protein kinase(MAPK) pathway through phosphorylation of GSK3 by kinases such asmitogen activated protein kinase activated protein kinase 1 (MAPKAP-K1or RSK). These data suggest that GSK3 activity may be modulated bymitogenic, insulin and/or amino acid stimulii.

It has also been shown that GSK3β is a key component in the vertebrateWnt signalling pathway. This biochemical pathway has been shown to becritical for normal embryonic development and regulates cellproliferation in normal tissues. GSK3 becomes inhibited in response toWnt stimulii. This can lead to the de-phosphorylation of GSK3 substratessuch as Axin, the adenomatous polyposis coli (APC) gene product andβ-catenin. Aberrant regulation of the Wnt pathway has been associatedwith many cancers. Mutations in APC, and/or β-catenin, are common incolorectal cancer and other tumours. β-catenin has also been shown to beof importance in cell adhesion. Thus GSK3 may also modulate cellularadhesion processes to some degree. Apart from the biochemical pathwaysalready described there are also data implicating GSK3 in the regulationof cell division via phosphorylation of cyclin-D1, in thephosphorylation of transcription factors such as c-Jun, CCAAT/enhancerbinding protein α (C/EBPα), c-Myc and/or other substrates such asNuclear Factor of Activated T-cells (NFATc), Heat Shock Factor-1 (HSF-1)and the c-AMP response element binding protein (CREB). GSK3 also appearsto play a role, albeit tissue specific, in regulating cellularapoptosis. The role of GSK3 in modulating cellular apoptosis, via apro-apoptotic mechanism, may be of particular relevance to medicalconditions in which neuronal apoptosis can occur. Examples of these arehead trauma, stroke, epilepsy, Alzheimer's and motor neuron diseases,progressive supranuclear palsy, corticobasal degeneration, and Pick'sdisease. In vitro it has been shown that GSK3 is able tohyper-phosphorylate the microtubule associated protein Tau.Hyperphosphorylation of Tau disrupts its normal binding to microtubulesand may also lead to the formation of intra-cellular Tau filaments. Itis believed that the progressive accumulation of these filaments leadsto eventual neuronal dysfunction and degeneration. Inhibition of Tauphosphorylation, through inhibition of GSK3, may thus provide a means oflimiting and/or preventing neurodegenerative effects.

Diffuse Large B-Cell Lymphomas (DLBCL)

Cell cycle progression is regulated by the combined action of cyclins,cyclin-dependent kinases (CDKs), and CDK-inhibitors (CDKi), which arenegative cell cycle regulators. p27KIP1 is a CDKi key in cell cycleregulation, whose degradation is required for G1/S transition. In spiteof the absence of p27KIP1 expression in proliferating lymphocytes, someaggressive B-cell lymphomas have been reported to show an anomalousp27KIP1 staining. An abnormally high expression of p27KIP1 was found inlymphomas of this type. Analysis of the clinical relevance of thesefindings showed that a high level of p27KIP1 expression in this type oftumour is an adverse prognostic marker, in both univariate andmultivariate analysis. These results show that there is abnormal p27KIP1expression in Diffuse Large B-cell Lymphomas (DLBCL), with adverseclinical significance, suggesting that this anomalous p27KIP1 proteinmay be rendered non-functional through interaction with other cell cycleregulator proteins. (Br. J. Cancer. 1999 July; 80(9):1427-34. p27KIP1 isabnormally expressed in Diffuse Large B-cell Lymphomas and is associatedwith an adverse clinical outcome. Saez A, Sanchez E, Sanchez-Beato M,Cruz M A, Chacon I, Munoz E, Camacho F I, Martinez-Montero J C, MollejoM, Garcia J F, Piris M A. Department of Pathology, Virgen de la SaludHospital, Toledo, Spain.)

Chronic Lymphocytic Leukemia

B-Cell chronic lymphocytic leukaemia (CLL) is the most common leukaemiain the Western hemisphere, with approximately 10,000 new cases diagnosedeach year (Parker S L, Tong T, Bolden S, Wingo P A: Cancer statistics,1997. Ca. Cancer. J. Clin. 47:5, (1997)). Relative to other forms ofleukaemia, the overall prognosis of CLL is good, with even the mostadvanced stage patients having a median survival of 3 years.

The addition of fludarabine as initial therapy for symptomatic CLLpatients has led to a higher rate of complete responses (27% v 3%) andduration of progression-free survival (33 v 17 months) as compared withpreviously used alkylator-based therapies. Although attaining a completeclinical response after therapy is the initial step toward improvingsurvival in CLL, the majority of patients either do not attain completeremission or fail to respond to fludarabine. Furthermore, all patientswith CLL treated with fludarabine eventually relapse, making its role asa single agent purely palliative (Rai K R, Peterson B, Elias L, ShepherdL, Hines J, Nelson D, Cheson B, Kolitz J, Schiffer C A: A randomizedcomparison of fludarabine and chlorambucil for patients with previouslyuntreated chronic lymphocytic leukemia. A CALGB SWOG, CTG/NCI-C and ECOGInter-Group Study. Blood 88:141a, 1996 (abstr 552, suppl 1). Therefore,identifying new agents with novel mechanisms of action that complementfludarabine's cytotoxicity and abrogate the resistance induced byintrinsic CLL drug-resistance factors will be necessary if furtheradvances in the therapy of this disease are to be realized.

The most extensively studied, uniformly predictive factor for poorresponse to therapy and inferior survival in CLL patients is aberrantp53 function, as characterized by point mutations or chromosome 17p13deletions. Indeed, virtually no responses to either alkylator or purineanalog therapy have been documented in multiple single institution caseseries for those CLL patients with abnormal p53 function. Introductionof a therapeutic agent that has the ability to overcome the drugresistance associated with p53 mutation in CLL would potentially be amajor advance for the treatment of the disease.

Flavopiridol and CYC 202, inhibitors of cyclin-dependent kinases inducein vitro apoptosis of malignant cells from B-cell chronic lymphocyticleukemia (B-CLL).

Flavopiridol exposure results in the stimulation of caspase 3 activityand in caspase-dependent cleavage of p27(kip1), a negative regulator ofthe cell cycle, which is overexpressed in B-CLL (Blood. 1998 Nov. 15;92(10):3804-16 Flavopiridol induces apoptosis in chronic lymphocyticleukemia cells via activation of caspase-3 without evidence of bcl-2modulation or dependence on functional p53. Byrd J C, Shinn C, WaselenkoJ K, Fuchs E J, Lehman T A, Nguyen P L, Flinn I W, Diehl L F, SausvilleE, Greyer M R).

Cytotoxic Compounds and Signalling Inhibitors

A wide variety of cytotoxic compounds and signalling inhibitors findapplication in the combinations of the invention, as described in detailbelow.

It is an object of the invention to provide therapeutic combinations ofpyrazole compounds that inhibit or modulate (in particular inhibit) theactivity of cyclin dependent kinases (CDK) and/or glycogen synthasekinase (e.g. GSK-3) with a cytotoxic compound or signalling inhibitor.Such combinations may have an advantageous efficacious effect againsttumour cell growth, in comparison with the respective effects shown bythe individual components of the combination.

Prior Art

WO 02/34721 from Du Pont discloses a class of indeno[1,2-c]pyrazol-4-ones as inhibitors of cyclin dependent kinases.

WO 01/81348 from Bristol Myers Squibb describes the use of 5-thio-,sulphinyl- and sulphonylpyrazolo[3,4-b]-pyridines as cyclin dependentkinase inhibitors.

WO 00/62778 also from Bristol Myers Squibb discloses a class of proteintyrosine kinase inhibitors.

WO 01/72745A1 from Cyclacel describes 2-substituted4-heteroaryl-pyrimidines and their preparation, pharmaceuticalcompositions containing them and their use as inhibitors ofcyclin-dependant kinases (CDKs) and hence their use in the treatment ofproliferative disorders such as cancer, leukaemia, psoriasis and thelike.

WO 99/21845 from Agouron describes 4-aminothiazole derivatives forinhibiting cyclin-dependent kinases (CDKs), such as CDK1, CDK2, CDK4,and CDK6. The invention is also directed to the therapeutic orprophylactic use of pharmaceutical compositions containing suchcompounds and to methods of treating malignancies and other disorders byadministering effective amounts of such compounds.

WO 01/53274 from Agouron discloses as CDK kinase inhibitors a class ofcompounds which can comprise an amide-substituted benzene ring linked toan N-containing heterocyclic group.

WO 01/98290 (Pharmacia & Upjohn) discloses a class of3-aminocarbonyl-2-carboxamido thiophene derivatives as protein kinaseinhibitors.

WO 01/53268 and WO 01/02369 from Agouron disclose compounds that mediateor inhibit cell proliferation through the inhibition of protein kinasessuch as cyclin dependent kinase or tyrosine kinase. The Agouroncompounds have an aryl or heteroaryl ring attached directly or though aCH═CH or CH═N group to the 3-position of an indazole ring.

WO 00/39108 and WO 02/00651 (both to Du Pont Pharmaceuticals) describeheterocyclic compounds that are inhibitors of trypsin-like serineprotease enzymes, especially factor Xa and thrombin. The compounds arestated to be useful as anticoagulants or for the prevention ofthromboembolic disorders.

US 2002/0091116 (Zhu et al.), WO 01/19798 and WO 01/64642 each disclosediverse groups of heterocyclic compounds as inhibitors of Factor Xa.Some 1-substituted pyrazole carboxamides are disclosed and exemplified.

U.S. Pat. No. 6,127,382, WO 01/70668, WO 00/68191, WO 97/48672, WO97/19052 and WO 97/19062 (all to Allergan) each describe compoundshaving retinoid-like activity for use in the treatment of varioushyperproliferative diseases including cancers.

WO 02/070510 (Bayer) describes a class of amino-dicarboxylic acidcompounds for use in the treatment of cardiovascular diseases. Althoughpyrazoles are mentioned generically, there are no specific examples ofpyrazoles in this document.

WO 97/03071 (Knoll A G) discloses a class of heterocyclyl-carboxamidederivatives for use in the treatment of central nervous systemdisorders. Pyrazoles are mentioned generally as examples of heterocyclicgroups but no specific pyrazole compounds are disclosed or exemplified.

WO 97/40017 (Novo Nordisk) describes compounds that are modulators ofprotein tyrosine phosphatases.

WO 03/020217 (Univ. Connecticut) discloses a class of pyrazole3-carboxamides as cannabinoid receptor modulators for treatingneurological conditions. It is stated (page 15) that the compounds canbe used in cancer chemotherapy but it is not made clear whether thecompounds are active as anti-cancer agents or whether they areadministered for other purposes.

WO 01/58869 (Bristol Myers Squibb) discloses cannabinoid receptormodulators that can be used inter alia to treat a variety of diseases.The main use envisaged is the treatment of respiratory diseases,although reference is made to the treatment of cancer.

WO 01/02385 (Aventis Crop Science) discloses1-(quinoline-4-yl)-1H-pyrazole derivatives as fungicides.1-Unsubstituted pyrazoles are disclosed as synthetic intermediates.

WO 2004/039795 (Fujisawa) discloses amides containing a 1-substitutedpyrazole group as inhibitors of apolipoprotein B secretion. Thecompounds are stated to be useful in treating such conditions ashyperlipidemia.

WO 2004/000318 (Cellular Genomics) discloses various amino-substitutedmonocycles as kinase modulators. None of the exemplified compounds arepyrazoles.

SUMMARY OF THE INVENTION

The invention provides combinations of (i) a cytotoxic compound orsignalling inhibitor; or (ii) an ancillary agent; or (iii) two or morefurther anti-cancer agents; with pyrazole compounds that have cyclindependent kinase inhibiting or modulating activity, wherein thecombinations have efficacy against abnormal cell growth. The inventionfurther provides combinations as described above which are furthercombined with other classes of therapeutic agents or treatments that maybe administered together (whether concurrently or at different timeintervals) as described in more detail hereinafter.

Thus, for example, it is envisaged that the combinations of theinvention will be useful in alleviating or reducing the incidence ofcancer.

Accordingly, in one aspect, the invention provides a combinationcomprising or consisting essentially of (i) a cytotoxic compound orsignalling inhibitor; or (ii) an ancillary agent; or (iii) two or morefurther anti-cancer agents; and a compound having the formula (0):

or salts or tautomers or N-oxides or solvates thereof;

wherein

-   -   X is a group R¹-A-NR⁴- or a 5- or 6-membered carbocyclic or        heterocyclic ring;    -   A is a bond, SO₂, C═O, NR^(g)(C═O) or O(C═O) wherein R^(g) is        hydrogen or C₁₋₄ hydrocarbyl optionally substituted by hydroxy        or C₁₋₄ alkoxy;    -   Y is a bond or an alkylene chain of 1, 2 or 3 carbon atoms in        length;    -   R¹ is hydrogen; a carbocyclic or heterocyclic group having from        3 to 12 ring members; or a C₁₋₈ hydrocarbyl group optionally        substituted by one or more substituents selected from halogen        (e.g. fluorine), hydroxy, C₁₋₄ hydrocarbyloxy, amino, mono- or        di-C₁₋₄ hydrocarbylamino, and carbocyclic or heterocyclic groups        having from 3 to 12 ring members, and wherein 1 or 2 of the        carbon atoms of the hydrocarbyl group may optionally be replaced        by an atom or group selected from O, S, NH, SO, SO₂;    -   R² is hydrogen; halogen; C₁₋₄ alkoxy (e.g. methoxy); or a C₁₋₄        hydrocarbyl group optionally substituted by halogen (e.g.        fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy);    -   R³ is selected from hydrogen and carbocyclic and heterocyclic        groups having from 3 to 12 ring members; and    -   R⁴ is hydrogen or a C₁₋₄ hydrocarbyl group optionally        substituted by halogen (e.g. fluorine), hydroxyl or C₁₋₄ alkoxy        (e.g. methoxy).

In one embodiment, the invention provides a combination of (i) acytotoxic compound or signalling inhibitor; or (ii) an ancillary agent;or (iii) two or more further anti-cancer agents; and a compound havingthe formula (I⁰):

or salts or tautomers or N-oxides or solvates thereof;

wherein

-   -   X is a group R¹-A-NR⁴- or a 5- or 6-membered carbocyclic or        heterocyclic ring;    -   A is a bond, C=O, NR^(g)(C═O) or O(C═O) wherein R^(g) is        hydrogen or C₁₋₄ hydrocarbyl optionally substituted by hydroxy        or C₁₋₄ alkoxy;    -   Y is a bond or an alkylene chain of 1, 2 or 3 carbon atoms in        length;    -   R¹ is hydrogen; a carbocyclic or heterocyclic group having from        3 to 12 ring members; or a C₁₋₈ hydrocarbyl group optionally        substituted by one or more substituents selected from halogen        (e.g. fluorine), hydroxy, C₁₋₄ hydrocarbyloxy, amino, mono- or        di-C₁₋₄ hydrocarbylamino, and carbocyclic or heterocyclic groups        having from 3 to 12 ring members, and wherein 1 or 2 of the        carbon atoms of the hydrocarbyl group may optionally be replaced        by an atom or group selected from O, S, NH, SO, SO₂;    -   R² is hydrogen; halogen; C₁₋₄ alkoxy (e.g. methoxy); or a C₁₋₄        hydrocarbyl group optionally substituted by halogen (e.g.        fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy);    -   R³ is selected from hydrogen and carbocyclic and heterocyclic        groups having from 3 to 12 ring members; and    -   R⁴ is hydrogen or a C₁₋₄ hydrocarbyl group optionally        substituted by halogen (e.g. fluorine), hydroxyl or C₁₋₄ alkoxy        (e.g. methoxy).

In a further embodiment, the invention provides a combination of (i) acytotoxic compound or signalling inhibitor; or (ii) an ancillary agent;or (iii) two or more further anti-cancer agents; and a compound havingthe formula (I):

or salts or tautomers or N-oxides or solvates thereof;

wherein

-   -   X is a group R¹-A-NR⁴—;    -   A is a bond, C═O, NR^(g)(C═O) or O(C═O) wherein R^(g) is        hydrogen or C₁₋₄ hydrocarbyl optionally substituted by hydroxy        or C₁₋₄ alkoxy;    -   Y is a bond or an alkylene chain of 1, 2 or 3 carbon atoms in        length;    -   R¹ is hydrogen; a carbocyclic or heterocyclic group having from        3 to 12 ring members; or a C₁₋₈ hydrocarbyl group optionally        substituted by one or more substituents selected from halogen        (e.g. fluorine), hydroxy, C₁₋₄ hydrocarbyloxy, amino, mono- or        di-C₁₋₄ hydrocarbylamino, and carbocyclic or heterocyclic groups        having from 3 to 12 ring members, and wherein 1 or 2 of the        carbon atoms of the hydrocarbyl group may optionally be replaced        by an atom or group selected from O, S, NH, SO, SO₂;    -   R² is hydrogen; halogen; C₁₋₄ alkoxy (e.g. methoxy); or a C₁₋₄        hydrocarbyl group optionally substituted by halogen (e.g.        fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy);    -   R³ is selected from hydrogen and carbocyclic and heterocyclic        groups having from 3 to 12 ring members; and    -   R⁴ is hydrogen or a C₁₋₄ hydrocarbyl group optionally        substituted by halogen (e.g. fluorine), hydroxyl or C₁₋₄ alkoxy        (e.g. methoxy).

Any one or more of the following optional provisos, in any combination,may apply to the compounds of formulae (0), (I⁰), (I) and sub-groupsthereof:

(a-i) When A is a bond and Y—R³ is an alkyl, cycloalkyl, optionallysubstituted phenyl or optionally substituted phenylalkyl, then R¹ isother than a substituted or unsubstituted dihydronaphthalene,dihydrochroman, dihydrothiochroman, tetrahydroquinoline ortetrahydrobenzfuranyl group.

(a-ii) X and R³ are each other than a moiety containing a maleimidegroup wherein the maleimide group has nitrogen atoms attached to the3-and 4-positions thereof.

(a-iii) R¹ is other than a moiety containing a purine nucleoside group.

(a-iv) X and R³ are each other than a moiety containing acyclobutene-1,2-dione group wherein the cyclobutene-1,2-dione group hasnitrogen atoms attached to the 3-and 4-positions thereof.

(a-v) R³ is other than a moiety containing a 4-monosubsituted or4,5-disubstituted 2-pyridyl or 2-pyrimidinyl group or a5-monosubstituted or 5,6-disubstituted 1,2,4-triazin-3-yl or3-pyridazinyl group.

(a-vi) X and R³ are each other than a moiety containing a substituted orunsubstituted pyrazol-3-ylamine group linked to a substituted orunsubstituted pyridine, diazine or triazine group.

(a-vii) When A is C═O and Y—R³ is an alkyl, cycloalkyl, optionallysubstituted phenyl or optionally substituted phenylalkyl group, then R¹is other than a substituted or unsubstituted tetrahydronaphthalene,tetrahydroquinolinyl, tetrahydrochromanyl or tetrahydrothiochromanylgroup.

(a-viii) When R³ is H and A is a bond, R¹ is other than a moietycontaining a bis-aryl, bis-heteroaryl or aryl heteroaryl group.

(a-ix) R³ is other than a moiety containing a1,2,8,8a-tetrahydro-7-methyl-cyclopropa[c]pyrrolo[3,2,e]indole-4-(5H)-onegroup.

(a-x) When Y is a bond, R³ is hydrogen, A is CO and R¹ is a substitutedphenyl group, each substituent on the phenyl group is other than a groupCH₂—P(O)R^(x)R^(y) where R^(x) and R^(y) are each selected from alkoxyand phenyl groups.

(a-xi) X is other than4-(tert-butyloxycarbonylamino)-3-methylimidazol-2-ylcarbonylamino.

In another embodiment, the invention provides a combination of (i) acytotoxic compound or signalling inhibitor; or (ii) an ancillary agent;or (iii) two or more further anti-cancer agents; and a compound havingthe formula (Ia):

or salts or tautomers or N-oxides or solvates thereof;

wherein

-   -   X is a group R¹-A-NR⁴—;    -   A is a bond, C═O, NR^(g)(C═O) or O(C═O) wherein R^(g) is        hydrogen or C₁₋₄ hydrocarbyl optionally substituted by hydroxy        or C₁₋₄ alkoxy;    -   Y is a bond or an alkylene chain of 1, 2 or 3 carbon atoms in        length;    -   R¹ is a carbocyclic or heterocyclic group having from 3 to 12        ring members; or a C₁₋₈ hydrocarbyl group optionally substituted        by one or more substituents selected from fluorine, hydroxy,        C₁₋₄ hydrocarbyloxy, amino, mono- or di-C₁₋₄ hydrocarbylamino,        and carbocyclic or heterocyclic groups having from 3 to 12 ring        members, and wherein 1 or 2 of the carbon atoms of the        hydrocarbyl group may optionally be replaced by an atom or group        selected from O, S, NH, SO, SO₂;    -   R² is hydrogen; halogen; C₁₋₄ alkoxy (e.g. methoxy); or a C₁₋₄        hydrocarbyl group optionally substituted by halogen (e.g.        fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy);    -   R³ is selected from hydrogen and carbocyclic and heterocyclic        groups having from 3 to 12 ring members; and    -   R⁴ is hydrogen or a C₁₋₄ hydrocarbyl group optionally        substituted by halogen (e.g. fluorine), hydroxyl or C₁₋₄ alkoxy        (e.g. methoxy).

Any one or more of the following optional provisos, in any combination,may apply to the compounds of formula (Ia) and sub-groups thereof:

Provisos (a-i) to (a-xi) above.

(b-i) R³ is other than a bridged azabicyclo group.

(b-ii) When A is a bond, then R³ is other than a moiety containing anunsubstituted or substituted phenyl group having attached to an orthoposition thereof, a substituted or unsubstituted carbamoyl orthiocarbamoyl group.

(b-iii) When A is a bond, then R³ is other than a moiety containing anisoquinoline or quinoxaline group each having attached thereto asubstituted or unsubstituted piperidine or piperazine ring.

(b-iv) When A is a bond and R¹ is an alkyl group, then R³ is other thana moiety containing a thiatriazine group.

(b-v) When R¹ or R³ contain a moiety in which a heterocyclic ring havingan S(═O)₂ ring member is fused to a carbocyclic ring, the saidcarbocyclic ring is other than a substituted or unsubstituted benzenering

(b-vi) When A is a bond, R¹ is other than an arylalkyl, heteroarylalkylor piperidinylalkyl group each having attached thereto a substituentselected from cyano, and substituted or unsubstituted amino, aminoalkyl,amidine, guanidine, and carbamoyl groups.

(b-vii) When X is a group R¹-A-NR⁴—, A is a bond and R¹ is anon-aromatic group, then R³ is other than a six membered monocyclic arylor heteroaryl group linked directly to a 5,6-fused bicyclic heteroarylgroup.

In another embodiment, the invention provides a combination of (i) acytotoxic compound or signalling inhibitor; or (ii) an ancillary agent;or (iii) two or more further anti-cancer agents; and a compound of theformula (Ib):

or salts or tautomers or N-oxides or solvates thereof;

wherein

-   -   X is a group R¹-A-NR⁴—;    -   A is a bond, C═O, NR^(g)(C═O) or O(C═O) wherein R^(g) is        hydrogen or C₁₋₄ hydrocarbyl optionally substituted by hydroxy        or C₁₋₄ alkoxy;    -   Y is a bond or an alkylene chain of 1, 2 or 3 carbon atoms in        length;    -   R¹ is a carbocyclic or heterocyclic group having from 3 to 12        ring members; or a C₁₋₈ hydrocarbyl group optionally substituted        by one or more substituents selected from fluorine, hydroxy,        C₁₋₄ hydrocarbyloxy, amino, mono- or di-C₁₋₄ hydrocarbylamino,        and carbocyclic or heterocyclic groups having from 3 to 12 ring        members, and wherein 1 or 2 of the carbon atoms of the        hydrocarbyl group may optionally be replaced by an atom or group        selected from O, S, NH, SO, SO₂;    -   R² is hydrogen; halogen; C₁₋₄ alkoxy (e.g. methoxy); or a C₁₋₄        hydrocarbyl group optionally substituted by halogen (e.g.        fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy);    -   R³ is selected from carbocyclic and heterocyclic groups having        from 3 to 12 ring members; and    -   R⁴ is hydrogen or a C₁₋₄ hydrocarbyl group optionally        substituted by halogen (e.g. fluorine), hydroxyl or C₁₋₄ alkoxy        (e.g. methoxy).

Any one or more of the following optional provisos, in any combination,may apply to the compounds of formula (Ib) and sub-groups thereof:

Provisos (a-i) to (a-vii), (a-ix) and (a-xi).

Provisos (b-i) to (b-vii).

(c-i) When A is a bond, R¹ is other than a substituted arylalkyl,heteroarylalkyl or piperidinylalkyl group.

(c-ii) When X is an amino or alkylamino group and Y is a bond, R³ isother than a disubstituted thiazolyl group wherein one of thesubstituents is selected from cyano and fluoroalkyl.

The reference in proviso (a-iii) to a purine nucleoside group refers tosubstituted and unsubstituted purine groups having attached thereto amonosaccharide group (e.g. a pentose or hexose) or a derivative of amonosaccharide group, for example a deoxy monosaccharide group or asubstituted monosaccharide group.

The reference in proviso (b-i) to a bridged azabicyclo group refers tobicycloalkane bridged ring systems in which one of the carbon atoms ofthe bicycloalkane has been replaced by a nitrogen atom. In bridged ringsystems, two rings share more than two atoms, see for example AdvancedOrganic Chemistry, by Jerry March, 4^(th) Edition, Wiley Interscience,pages 131-133, 1992.

The provisos (a-i) to (a-x), (b-i) to (b-vii), (c-i) and (c-ii) informulae (I), (Ia) and (Ib) above refer to the disclosures in thefollowing prior art documents.

(a-i) US 2003/0166932, U.S. Pat. No. 6,127,382, U.S. Pat. No. 6,093,838

(a-ii) WO 03/031440

(a-iii) WO 03/014137

(a-iv) WO 02/083624

(a-v) WO 02/064586

(a-vi) WO 02/22608, WO 02/22605, WO 02/22603 & WO 02/22601

(a-vii) WO 97/48672, WO 97/19052

(a-viii) WO 00/06169

(a-ix) U.S. Pat. No. 5,502,068

(a-x) JP 07188269

(b-i) WO 03/040147

(b-ii) WO 01/70671

(b-iii) WO 01/32626

(b-iv) WO 98/08845

(b-v) WO 00/59902

(b-vi) U.S. Pat. No. 6,020,357, WO 99/32454 & WO 98/28269

(b-vii) WO 2004/012736

(c-i) U.S. Pat. No. 6,020,357, WO 99/32454 & WO 98/28269

(c-ii) US 2004/0082629

Any one or more of the foregoing optional provisos, (a-i) to (a-xi),(b-i) to (b-vii), (c-i) and (c-ii) in any combination, may also apply tothe compounds of formulae (Ib), (II), (III), (IV), (IVa), (Va), (Vb),(Via), (VIb), (VII) or (VIII) and sub-groups thereof or salts ortautomers or N-oxides or solvates thereof as defined herein.

In the following aspects and embodiments of the invention, references to“a combination according to the invention” refer to the combination of(i) a cytotoxic compound or signalling inhibitor; or (ii) an ancillaryagent; or (iii) two or more further anti-cancer agents; and a compoundof formula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),(Vb), (VIa), (VIb), (VII) or (VIII). In this section, as in all othersections of this application, unless the context indicates otherwise,references to a compound of formula (0), (I⁰), (I), (Ia), (Ib), (II),(III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) includesall other subgroups as defined herein. The term ‘subgroups’ includes allpreferences, examples and particular compounds defined herein.

Moreover, a reference to a compound of formula (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof includes ionic, salt, solvate, isomers,tautomers, N-oxides, ester, prodrugs, isotopes and protected formsthereof, as discussed below. Preferably, the salts or tautomers orisomers or N-oxides or solvates thereof. More preferably, the salts ortautomers or N-oxides or solvates thereof.

The invention also provides:

-   -   A combination according to the invention for use in alleviating        or reducing the incidence of a disease or condition comprising        or arising from abnormal cell growth in a mammal.    -   A combination of the invention for use in the prophylaxis or        treatment of a disease state or condition mediated by a cyclin        dependent kinase or glycogen synthase kinase-3.    -   A method for the prophylaxis or treatment of a disease state or        condition mediated by a cyclin dependent kinase or glycogen        synthase kinase-3, which method comprises administering to a        subject in need thereof a combination of the invention.    -   A method for alleviating or reducing the incidence of a disease        state or condition mediated by a cyclin dependent kinase or        glycogen synthase kinase-3, which method comprises administering        to a subject in need thereof a combination of the invention.    -   A method for alleviating or reducing the incidence of a disease        or condition comprising or arising from abnormal cell growth in        a mammal, which method comprises administering to the mammal a        combination according to the invention in an amount effective in        inhibiting abnormal cell growth.    -   A method for treating a disease or condition comprising or        arising from abnormal cell growth in a mammal, which method        comprises administering to the mammal a combination according to        the invention in an amount effective in inhibiting abnormal cell        growth.    -   A combination according to the invention for use in inhibiting        tumour growth in a mammal.    -   A method of inhibiting tumour growth in a mammal, which method        comprises administering to the mammal an effective tumour        growth-inhibiting amount of a combination according to the        invention.    -   A combination according to the invention for use in inhibiting        the growth of tumour cells.    -   A method of inhibiting the growth of tumour cells, which method        comprises contacting the tumour cells with administering to the        mammal an effective tumour cell growth-inhibiting amount of a        combination according to the invention.    -   A pharmaceutical composition comprising a combination according        to the invention and a pharmaceutically acceptable carrier.    -   A combination according to the invention for use in medicine.    -   The use of a combination according to the invention, for the        manufacture of a medicament for the prophylaxis or treatment of        any one of the disease states or conditions disclosed herein.    -   A method for the treatment or prophylaxis of any one of the        disease states or conditions disclosed herein, which method        comprises administering to a patient (e.g. a patient in need        thereof) a combination according to the invention.    -   A method for alleviating or reducing the incidence of a disease        state or condition disclosed herein, which method comprises        administering to a patient (e,g, a patient in need thereof) a        combination according to the invention.    -   A method for the diagnosis and treatment of a cancer in a        mammalian patient, which method comprises (a) screening a        patient to determine whether a cancer from which the patient is        or may be suffering is one which would be susceptible to        treatment with a compound having activity against cyclin        dependent kinases and (i) a cytotoxic compound or signalling        inhibitor; or (ii) an ancillary agent; or (iii) two or more        further anti-cancer agents; and (b) where it is indicated that        the disease or condition from which the patient is thus        susceptible, thereafter administering to the patient a        combination according to the invention.    -   The use of a combination according to the invention for the        manufacture of a medicament for the treatment or prophylaxis of        a cancer in a patient who has been screened and has been        determined as suffering from, or being at risk of suffering        from, a cancer which would be susceptible to treatment with a        combination of (i) a cytotoxic compound or signalling inhibitor;        or (ii) an ancillary agent; or (iii) two or more further        anti-cancer agents; and a compound having activity against        cyclin dependent kinase.    -   A method for treating a cancer in a patient comprising        administration of a combination according to the invention to        said patient in an amount and in a schedule of administration        that is therapeutically efficaceous in the treatment of said        cancer.    -   A method for preventing, treating or managing cancer in a        patient in need thereof, said method comprising administering to        said patient a prophylactically or therapeutically effective        amount of a combination according to the invention.    -   The use of a combination according to the invention for the        manufacture of a medicament for use in the production of an        anti-cancer effect in a warm-blooded animal such as a human.    -   A kit comprising a combination according to the invention.    -   A method for the treatment of a cancer in a warm-blooded animal        such as a human, which comprises administering to said animal an        effective amount of (i) a cytotoxic compound or signalling        inhibitor; or (ii) an ancillary agent; or (iii) two or more        further anti-cancer agents; sequentially e.g. before or after,        or simultaneously with an effective amount of a compound of the        formula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa),        (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof        as defined herein.    -   A pharmaceutical kit for anticancer therapy comprising (i) a        cytotoxic compound or signalling inhibitor; or (ii) an ancillary        agent; or (iii) two or more further anti-cancer agents; in        dosage form and a compound of the formula (0), (I⁰), (I), (Ia),        (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (Via), (VIb), (VII)        or (VIII) and sub-groups thereof as defined herein, also in        dosage form (e.g. wherein the dosage forms are packaged together        in common outer packaging).    -   A method of combination cancer therapy in a mammal comprising        administering a therapeutically effective amount of (i) a        cytotoxic compound or signalling inhibitor; or (ii) an ancillary        agent; or (iii) two or more further anti-cancer agents; and a        therapeutically effective amount of a compound of the formula        (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),        (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof as        defined herein.    -   A compound of the formula (0), (I⁰), (I), (Ia), (Ib), (II),        (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII)        and sub-groups thereof as defined herein for use in combination        therapy with (i) a cytotoxic compound or signalling inhibitor;        or (ii) an ancillary agent; or (iii) two or more further        anti-cancer agents; to alleviate or reduce the incidence of a        disease or condition comprising or arising from abnormal cell        growth in a mammal.    -   A compound of the formula (0), (I⁰), (I), (Ia), (Ib), (II),        (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII)        and sub-groups thereof as defined herein for use in combination        therapy with (i) a cytotoxic compound or signalling inhibitor;        or (ii) an ancillary agent; or (iii) two or more further        anti-cancer agents; to inhibit tumour growth in a mammal.    -   A compound of the formula (0), (I⁰), (I), (Ia), (Ib), (II),        (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII)        and sub-groups thereof as defined herein for use in combination        therapy with (i) a cytotoxic compound or signalling inhibitor;        or (ii) an ancillary agent; or (iii) two or more further        anti-cancer agents; to prevent, treat or manage cancer in a        patient in need thereof.    -   A compound of the formula (0), (I⁰), (I), (Ia), (Ib), (II),        (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII)        and sub-groups thereof as defined herein for use in enhancing or        potentiating the response rate in a patient suffering from a        cancer where the patient is being treated with (i) a cytotoxic        compound or signalling inhibitor; or (ii) an ancillary agent;        or (iii) two or more further anti-cancer agents.    -   A method of enhancing or potentiating the response rate in a        patient suffering from a cancer where the patient is being        treated with (i) a cytotoxic compound or signalling inhibitor;        or (ii) an ancillary agent; or (iii) two or more further        anti-cancer agents; which method comprises administering to the        patient, in combination with the (i) cytotoxic compound or        signalling inhibitor ; or (ii) an ancillary agent; or (iii) two        or more further anti-cancer agents; a compound of the formula        (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),        (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof as        defined herein.    -   The use of a combination according to the invention for the        manufacture of a medicament for any of the therapeutic uses as        defined herein.

In each of the foregoing uses, methods and other aspects of theinvention, as well as any aspects and embodiments of the invention asset out below, references to compounds of the formulae (0), (I⁰), (I),(Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein include within theirscope the salts or solvates or tautomers or N-oxides of the compounds.

The invention also provides the further combinations, uses, methods,compounds and processes as set out in the claims below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the percent control proliferation in A2780 cells forGemcitibine in the presence and absence of 0.3 μM Compound I addedconcurrently, using the example of the IC₅₀ response curves toGemcitibine.

FIG. 2 depicts the percent control proliferation in A2780 cells in thepresence and absence of either 0.01 uM Gemcitabine, 0.3 uM Compound I orboth added concurrently using the example of percentage controlproliferation following each treatment regimen.

FIG. 3 depicts the percent control proliferation in A2780 cells forPaclitaxel in the presence and absence of 0.3 μM Compound I addedconcurrently, using the example of the IC₅₀ response curve toPaclitaxel.

FIG. 4 depicts the percent control proliferation in A2780 cells forPaclitaxel in the presence and absence of either 0.003 uM Paclitaxel,0.3 μM Compound I or both added concurrently using the example ofpercentage control proliferation following each treatment regimen.

FIG. 5 depicts the percent control proliferation in A2780 cells forPaclitaxel in the absence of Compound I, and followed by addition of 0.3μM Compound I, using the example of the IC₅₀ response curve toPaclitaxel.

FIG. 6 depicts the percent control proliferation in A2780 cells forPaclitaxel in the absence of either 0.003 uM Paclitaxel, 0.3 uM CompoundI or a combination where Paclitaxel is added 24 h prior to Compound Iusing the example of percentage control proliferation following eachtreatment regimen.

FIG. 7 depicts the percent control proliferation in A2780 cells for 5-FUin the presence and absence of 0.15 μM Compound I added concurrently,using the example of the IC₅₀ response curves to 5-FU.

FIG. 8 depicts the percent control proliferation in A2780 cells for 5-FUin the presence and absence of either 1 uM 5-FU, 0.15 uM Compound I orboth added concurrently using the example of percentage controlproliferation following each treatment regimen.

FIG. 9 depicts the percent control proliferation in A2780 cells forIressa in the presence and absence of 0.2 μM Compound I added incombination therewith, using the example of the IC₅₀ response curves toIressa.

FIG. 10 depicts the percent control proliferation in A2780 cells forIressa in the presence and absence of either 3.8 uM Iressa, 0.2 uMCompound I or both added concurrently using the example of percentagecontrol proliferation following each treatment regimen.

FIG. 11 depicts the percent control proliferation in HT29 cells forcamptothecin in the absence of Compound I, and 0.1 μM Compound I addedprior to camptothecin, using the example of the IC₅₀ response curve tocamptothecin.

FIG. 12 depicts the percent control proliferation in HT29 cells forcamptothecin in the absence of either 0.04 uM camptothecin, 1 uMCompound I or a combination where Compound I is added 24 h prior tocamptothecin using the example of percentage control proliferationfollowing each treatment regimen.

FIG. 13 depicts the percent control proliferation in A2780 cells forVinblastine in the presence and absence of 0.3 μM Compound I addedconcurrently, using the example of the IC₅₀ response curves toVinblastine.

FIG. 14 depicts the percent control proliferation in A2780 cells forVinblastine in the presence and absence of either 0.03 uM Vinblastine,0.3 uM Compound I or both added concurrently using the example ofpercentage control proliferation following each treatment regimen.

FIG. 15 depicts the percent control proliferation in A2780 cells forCisplatin in the presence and absence of 0.3 μM Compound I addedconcurrently, using the example of the IC₅₀ response curves toCisplatin.

FIG. 16 depicts the percent control proliferation in A2780 cells forCisplatin in the presence and absence of either 1 uM cisplatin, 0.3 uMCompound I or both added concurrently using the example of percentagecontrol proliferation following each treatment regimen.

FIG. 17 depicts the percent control proliferation in A2780 cells forEtoposide in the presence and absence of 0.075 μM Compound I addedconcurrently, using the example of the IC₅₀ response curves toEtoposide.

FIG. 18 depicts the percent control proliferation in A2780 cells forEtoposide in the presence and absence of either 0.03 uM Etoposide, 0.075uM Compound I or both added concurrently using the example of percentagecontrol proliferation following each treatment regimen.

FIG. 19 depicts the crystal structure of4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate, which was solved as describedbelow.

FIG. 20 depicts a graphical representation of the structure of4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate, generated by X-ray diffractionstudy.

DETAILED DESCRIPTION OF THE INVENTION

General Preferences and Definitions

As used herein, the term “modulation”, as applied to the activity ofcyclin dependent kinase (CDK) and glycogen synthase kinase (GSK, e.g.GSK-3), is intended to define a change in the level of biologicalactivity of the kinase(s). Thus, modulation encompasses physiologicalchanges which effect an increase or decrease in the relevant kinaseactivity. In the latter case, the modulation may be described as“inhibition”. The modulation may arise directly or indirectly, and maybe mediated by any mechanism and at any physiological level, includingfor example at the level of gene expression (including for exampletranscription, translation and/or post-translational modification), atthe level of expression of genes encoding regulatory elements which actdirectly or indirectly on the levels of cyclin dependent kinase (CDK)and/or glycogen synthase kinase-3 (GSK-3) activity, or at the level ofenzyme (e.g. cyclin dependent kinase (CDK) and/or glycogen synthasekinase-3 (GSK-3)) activity (for example by allosteric mechanisms,competitive inhibition, active-site inactivation, perturbation offeedback inhibitory pathways etc.). Thus, modulation may implyelevated/suppressed expression or over- or under-expression of thecyclin dependent kinase (CDK) and/or glycogen synthase kinase-3 (GSK-3),including gene amplification (i.e. multiple gene copies) and/orincreased or decreased expression by a transcriptional effect, as wellas hyper- (or hypo-)activity and (de)activation of the cyclin dependentkinase (CDK) and/or glycogen synthase kinase-3 (GSK-3) (including(de)activation) by mutation(s). The terms “modulated” and “modulate” areto be interpreted accordingly.

As used herein, the term “mediated”, as used e.g. in conjunction withthe cyclin dependent kinases (CDK) and/or glycogen synthase kinase-3(GSK-3) as described herein (and applied for example to variousphysiological processes, diseases, states, conditions, therapies,treatments or interventions) is intended to operate limitatively so thatthe various processes, diseases, states, conditions, treatments andinterventions to which the term is applied are those in which cyclindependent kinase (CDK) and/or glycogen synthase kinase-3 (GSK-3) plays abiological role. In cases where the term is applied to a disease, stateor condition, the biological role played by cyclin dependent kinase(CDK) and/or glycogen synthase kinase-3 (GSK-3) may be direct orindirect and may be necessary and/or sufficient for the manifestation ofthe symptoms of the disease, state or condition (or its aetiology orprogression). Thus, cyclin dependent kinase (CDK) and/or glycogensynthase kinase-3 (GSK-3) activity (and in particular aberrant levels ofcyclin dependent kinase (CDK) and/or glycogen synthase kinase-3 (GSK-3)activity, e.g. cyclin dependent kinases (CDK) and/or glycogen synthasekinase-3 (GSK-3) over-expression) need not necessarily be the proximalcause of the disease, state or condition: rather, it is contemplatedthat the CDK- and/or GSK- (e.g. GSK-3-) mediated diseases, states orconditions include those having multifactorial aetiologies and complexprogressions in which CDK and/or GSK-3 is only partially involved. Incases where the term is applied to treatment, prophylaxis orintervention (e.g. in the “CDK-mediated treatments” and “GSK-3-mediatedprophylaxis” of the invention), the role played by CDK and/or GSK-3 maybe direct or indirect and may be necessary and/or sufficient for theoperation of the treatment, prophylaxis or outcome of the intervention.

The term “intervention” is a term of art used herein to define anyagency which effects a physiological change at any level. Thus, theintervention may comprises the induction or repression of anyphysiological process, event, biochemical pathway orcellular/biochemical event. The interventions of the invention typicallyeffect (or contribute to) the therapy, treatment or prophylaxis of adisease or condition.

The combinations of the invention are combinations of a cytotoxiccompound or signalling inhibitor and a compound of the formulae (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof that produce atherapeutically efficacious effect.

The term ‘efficacious’ includes advantageous effects such as additivity,synergism, reduced side effects, reduced toxicity, increased time todisease progression, increased time of survival, sensitization orresensitization of one agent to another, or improved response rate.Advantageously, an efficacious effect may allow for lower doses of eachor either component to be administered to a patient, thereby decreasingthe toxicity of chemotherapy, whilst producing and/or maintaining thesame therapeutic effect.

A “synergistic” effect in the present context refers to a therapeuticeffect produced by the combination which is larger than the sum of thetherapeutic effects of the components of the combination when presentedindividually.

An “additive” effect in the present context refers to a therapeuticeffect produced by the combination which is larger than the therapeuticeffect of any of the components of the combination when presentedindividually.

The term “response rate” as used herein refers, in the case of a solidtumour, to the extent of reduction in the size of the tumour at a giventime point, for example 12 weeks. Thus, for example, a 50% response ratemeans a reduction in tumour size of 50%. References herein to a“clinical response” refer to response rates of 50% or greater. A“partial response” is defined herein as being a response rate of lessthan 50%.

As used herein, the term “combination”, as applied to two or morecompounds, may define material in which the two or more compounds areassociated. The terms “combined” and “combining” in this context are tobe interpreted accordingly.

The association of the two or more compounds in a combination may bephysical or non-physical. Examples of physically associated combinedcompounds include:

-   -   compositions (e.g. unitary formulations) comprising the two or        more compounds in admixture (for example within the same unit        dose);    -   compositions comprising material in which the two or more        compounds are chemically/physicochemically linked (for example        by crosslinking, molecular agglomeration or binding to a common        vehicle moiety);    -   compositions comprising material in which the two or more        compounds are chemically/physicochemically co-packaged (for        example, disposed on or within lipid vesicles, particles (e.g.        micro- or nanoparticles) or emulsion droplets);    -   pharmaceutical kits, pharmaceutical packs or patient packs in        which the two or more compounds are co-packaged or co-presented        (e.g. as part of an array of unit doses);

Examples of non-physically associated combined compounds include:

-   -   material (e.g. a non-unitary formulation) comprising at least        one of the two or more compounds together with instructions for        the extemporaneous association of the at least one compound to        form a physical association of the two or more compounds;    -   material (e.g. a non-unitary formulation) comprising at least        one of the two or more compounds together with instructions for        combination therapy with the two or more compounds;    -   material comprising at least one of the two or more compounds        together with instructions for administration to a patient        population in which the other(s) of the two or more compounds        have been (or are being) administered;    -   material comprising at least one of the two or more compounds in        an amount or in a form which is specifically adapted for use in        combination with the other(s) of the two or more compounds.

As used herein, the term “combination therapy” is intended to definetherapies which comprise the use of a combination of two or morecompounds (as defined above). Thus, references to “combination therapy”,“combinations” and the use of compounds “in combination” in thisapplication may refer to compounds that are administered as part of thesame overall treatment regimen. As such, the posology of each of the twoor more compounds may differ: each may be administered at the same timeor at different times. It will therefore be appreciated that thecompounds of the combination may be administered sequentially (e.g.before or after) or simultaneously, either in the same pharmaceuticalformulation (i.e. together), or in different pharmaceutical formulations(i.e. separately). Simultaneously in the same formulation is as aunitary formulation whereas simultaneously in different pharmaceuticalformulations is non-unitary. The posologies of each of the two or morecompounds in a combination therapy may also differ with respect to theroute of administration.

As used herein, the term “pharmaceutical kit” defines an array of one ormore unit doses of a pharmaceutical composition together with dosingmeans (e.g. measuring device) and/or delivery means (e.g. inhaler orsyringe), optionally all contained within common outer packaging. Inpharmaceutical kits comprising a combination of two or more compounds,the individual compounds may unitary or non-unitary formulations. Theunit dose(s) may be contained within a blister pack. The pharmaceuticalkit may optionally further comprise instructions for use.

As used herein, the term “pharmaceutical pack” defines an array of oneor more unit doses of a pharmaceutical composition, optionally containedwithin common outer packaging. In pharmaceutical packs comprising acombination of two or more compounds, the individual compounds mayunitary or non-unitary formulations. The unit dose(s) may be containedwithin a blister pack. The pharmaceutical pack may optionally furthercomprise instructions for use.

As used herein, the term “patient pack” defines a package, prescribed toa patient, which contains pharmaceutical compositions for the wholecourse of treatment. Patient packs usually contain one or more blisterpack(s). Patient packs have an advantage over traditional prescriptions,where a pharmacist divides a patient's supply of a pharmaceutical from abulk supply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in patientprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions.

The combinations of the invention may produce a therapeuticallyefficacious effect relative to the therapeutic effect of the individualcompounds when administered separately.

The following general preferences and definitions shall apply to each ofthe moieties X, Y, R^(g), R¹ to R⁴ and any sub-definition, sub-group orembodiment thereof, unless the context indicates otherwise.

In this specification, references to formula (I) include formulae (0),(I⁰), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb),(VII) or (VIII) and sub-groups, examples or embodiments of formulae (0),(I⁰), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb),(VII) or (VIII) unless the context indicates otherwise.

Thus for example, references to inter alia therapeutic uses,pharmaceutical formulations and processes for making compounds, wherethey refer to formula (I), are also to be taken as referring to formulae(0), (I⁰), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups, examples or embodiments offormulae (0), (I⁰), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb),(VIa), (VIb), (VII) or (VIII).

Similarly, where preferences, embodiments and examples are given forcompounds of the formula (I), they are also applicable to formulae (0),(I⁰), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb),(VII) or (VIII) and sub-groups, examples or embodiments of formulae (0),(I⁰), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb),(VII) or (VIII) unless the context requires otherwise.

References to “carbocyclic” and “heterocyclic” groups as used hereinshall, unless the context indicates otherwise, include both aromatic andnon-aromatic ring systems. Thus, for example, the term “carbocyclic andheterocyclic groups” includes within its scope aromatic, non-aromatic,unsaturated, partially saturated and fully saturated carbocyclic andheterocyclic ring systems. In general, such groups may be monocyclic orbicyclic and may contain, for example, 3 to 12 ring members, moreusually 5 to 10 ring members. Examples of monocyclic groups are groupscontaining 3, 4, 5, 6, 7, and 8 ring members, more usually 3 to 7, andpreferably 5 or 6 ring members. Examples of bicyclic groups are thosecontaining 8, 9, 10, 11 and 12 ring members, and more usually 9 or 10ring members.

The carbocyclic or heterocyclic groups can be aryl or heteroaryl groupshaving from 5 to 12 ring members, more usually from 5 to 10 ringmembers. The term “aryl” as used herein refers to a carbocyclic grouphaving aromatic character and the term “heteroaryl” is used herein todenote a heterocyclic group having aromatic character. The terms “aryl”and “heteroaryl” embrace polycyclic (e.g. bicyclic) ring systems whereinone or more rings are non-aromatic, provided that at least one ring isaromatic. In such polycyclic systems, the group may be attached by thearomatic ring, or by a non-aromatic ring. The aryl or heteroaryl groupscan be monocyclic or bicyclic groups and can be unsubstituted orsubstituted with one or more substituents, for example one or moregroups R¹⁰ as defined herein.

The term “non-aromatic group” embraces unsaturated ring systems withoutaromatic character, partially saturated and fully saturated carbocyclicand heterocyclic ring systems. The terms “unsaturated” and “partiallysaturated” refer to rings wherein the ring structure(s) contains atomssharing more than one valence bond i.e. the ring contains at least onemultiple bond e.g. a C=C, CC or N=C bond. The term “fully saturated”refers to rings where there are no multiple bonds between ring atoms.Saturated carbocyclic groups include cycloalkyl groups as defined below.Partially saturated carbocyclic groups include cycloalkenyl groups asdefined below, for example cyclopentenyl, cycloheptenyl andcyclooctenyl. A further example of a cycloalkenyl group is cyclohexenyl.

Examples of heteroaryl groups are monocyclic and bicyclic groupscontaining from five to twelve ring members, and more usually from fiveto ten ring members. The heteroaryl group can be, for example, a fivemembered or six membered monocyclic ring or a bicyclic structure formedfrom fused five and six membered rings or two fused six membered ringsor, by way of a further example, two fused five membered rings. Eachring may contain up to about four heteroatoms typically selected fromnitrogen, sulphur and oxygen. Typically the heteroaryl ring will containup to 4 heteroatoms, more typically up to 3 heteroatoms, more usually upto 2, for example a single heteroatom. In one embodiment, the heteroarylring contains at least one ring nitrogen atom.

The nitrogen atoms in the heteroaryl rings can be basic, as in the caseof an imidazole or pyridine, or essentially non-basic as in the case ofan indole or pyrrole nitrogen. In general the number of basic nitrogenatoms present in the heteroaryl group, including any amino groupsubstituents of the ring, will be less than five.

Examples of five membered heteroaryl groups include but are not limitedto pyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole,oxatriazole, isoxazole, thiazole, isothiazole, pyrazole, triazole andtetrazole groups.

Examples of six membered heteroaryl groups include but are not limitedto pyridine, pyrazine, pyridazine, pyrimidine and triazine.

A bicyclic heteroaryl group may be, for example, a group selected from:

-   -   a) a benzene ring fused to a 5- or 6-membered ring containing 1,        2 or 3 ring heteroatoms;    -   b) a pyridine ring fused to a 5- or 6-membered ring containing        1, 2 or 3 ring heteroatoms;    -   c) a pyrimidine ring fused to a 5- or 6-membered ring containing        1 or 2 ring heteroatoms;    -   d) a pyrrole ring fused to a a 5- or 6-membered ring containing        1, 2 or 3 ring heteroatoms;    -   e) a pyrazole ring fused to a a 5- or 6-membered ring containing        1 or 2 ring heteroatoms;    -   f) an imidazole ring fused to a 5- or 6-membered ring containing        1 or 2 ring heteroatoms;    -   g) an oxazole ring fused to a 5- or 6-membered ring containing 1        or 2 ring heteroatoms;    -   h) an isoxazole ring fused to a 5- or 6-membered ring containing        1 or 2 ring heteroatoms;    -   i) a thiazole ring fused to a 5- or 6-membered ring containing 1        or 2 ring heteroatoms;    -   j) an isothiazole ring fused to a 5- or 6-membered ring        containing 1 or 2 ring heteroatoms;    -   k) a thiophene ring fused to a 5- or 6-membered ring containing        1, 2 or 3 ring heteroatoms;    -   l) a furan ring fused to a 5- or 6-membered ring containing 1, 2        or 3 ring heteroatoms;    -   m) an oxazole ring fused to a 5- or 6-membered ring containing 1        or 2 ring heteroatoms;    -   n) an isoxazole ring fused to a 5- or 6-membered ring containing        1 or 2 ring heteroatoms;    -   o) a cyclohexyl ring fused to a 5- or 6-membered ring containing        1, 2 or 3 ring heteroatoms; and    -   p) a cyclopentyl ring fused to a 5- or 6-membered ring        containing 1, 2 or 3 ring heteroatoms.

Particular examples of bicyclic heteroaryl groups containing a fivemembered ring fused to another five membered ring include but are notlimited to imidazothiazole (e.g. imidazo[2,1-b]thiazole) andimidazoimidazole (e.g. imidazo[1,2-a]imidazole).

Particular examples of bicyclic heteroaryl groups containing a sixmembered ring fused to a five membered ring include but are not limitedto benzfuran, benzthiophene, benzimidazole, benzoxazole, isobenzoxazole,benzisoxazole, benzthiazole, benzisothiazole, isobenzofuran, indole,isoindole, indolizine, indoline, isoindoline, purine (e.g., adenine,guanine), indazole, pyrazolopyrimidine (e.g. pyrazolo[1,5-a]pyrimidine),triazolopyrimidine (e.g. [1,2,4]triazolo[1,5-a]pyrimidine), benzodioxoleand pyrazolopyridine (e.g. pyrazolo[1,5-a]pyridine) groups.

Particular examples of bicyclic heteroaryl groups containing two fusedsix membered rings include but are not limited to quinoline,isoquinoline, chroman, thiochroman, chromene, isochromene, chroman,isochroman, benzodioxan, quinolizine, benzoxazine, benzodiazine,pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine,naphthyridine and pteridine groups.

One sub-group of heteroaryl groups comprises pyridyl, pyrrolyl, furanyl,thienyl, imidazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl,thiazolyl, isothiazolyl, pyrazolyl, pyrazinyl, pyridazinyl, pyrimidinyl,triazinyl, triazolyl, tetrazolyl, quinolinyl, isoquinolinyl,benzfuranyl, benzthienyl, chromanyl, thiochromanyl, benzimidazolyl,benzoxazolyl, benzisoxazole, benzthiazolyl and benzisothiazole,isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl,isoindolinyl, purinyl (e.g., adenine, guanine), indazolyl,benzodioxolyl, chromenyl, isochromenyl, isochromanyl, benzodioxanyl,quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl andpteridinyl groups.

Examples of polycyclic aryl and heteroaryl groups containing an aromaticring and a non-aromatic ring include tetrahydronaphthalene,tetrahydroisoquinoline, tetrahydroquinoline, dihydrobenzthiene,dihydrobenzfuran, 2,3-dihydro-benzo[1,4]dioxine, benzo[1,3]dioxole,4,5,6,7-tetrahydrobenzofuran, indoline and indane groups.

Examples of carbocyclic aryl groups include phenyl, naphthyl, indenyl,and tetrahydronaphthyl groups.

Examples of non-aromatic heterocyclic groups include unsubstituted orsubstituted (by one or more groups R¹⁰) heterocyclic groups having from3 to 12 ring members, typically 4 to 12 ring members, and more usuallyfrom 5 to 10 ring members. Such groups can be monocyclic or bicyclic,for example, and typically have from 1 to 5 heteroatom ring members(more usually 1,2,3 or 4 heteroatom ring members) typically selectedfrom nitrogen, oxygen and sulphur.

When sulphur is present, it may, where the nature of the adjacent atomsand groups permits, exist as —S—, —S(O)— or —S(O)₂—.

The heterocylic groups can contain, for example, cyclic ether moieties(e.g. as in tetrahydrofuran and dioxane), cyclic thioether moieties(e.g. as in tetrahydrothiophene and dithiane), cyclic amine moieties(e.g. as in pyrrolidine), cyclic amide moieties (e.g. as inpyrrolidone), cyclic thioamides, cyclic thioesters, cyclic estermoieties (e.g. as in butyrolactone), cyclic sulphones (e.g. as insulpholane and sulpholene), cyclic sulphoxides, cyclic sulphonamides andcombinations thereof (e.g. morpholine and thiomorpholine and its S-oxideand S,S-dioxide). Further examples of heterocyclic groups are thosecontaining a cyclic urea moiety (e.g. as in imidazolidin-2-one),

In one sub-set of heterocyclic groups, the heterocyclic groups containcyclic ether moieties (e.g as in tetrahydrofuran and dioxane), cyclicthioether moieties (e.g. as in tetrahydrothiophene and dithiane), cyclicamine moieties (e.g. as in pyrrolidine), cyclic sulphones (e.g. as insulpholane and sulpholene), cyclic sulphoxides, cyclic sulphonamides andcombinations thereof (e.g. thiomorpholine).

Examples of monocyclic non-aromatic heterocyclic groups include 5-,6-and 7-membered monocyclic heterocyclic groups. Particular examplesinclude morpholine, piperidine (e.g. 1-piperidinyl, 2-piperidinyl,3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g. 1-pyrrolidinyl,2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone, pyran (2H-pyran or4H-pyran), dihydrothiophene, dihydropyran, dihydrofuran,dihydrothiazole, tetrahydrofuran, tetrahydrothiophene, dioxane,tetrahydropyran (e.g. 4-tetrahydro pyranyl), imidazoline,imidazolidinone, oxazoline, thiazoline, 2-pyrazoline, pyrazolidine,piperazine, and N-alkyl piperazines such as N-methyl piperazine. Furtherexamples include thiomorpholine and its S-oxide and S,S-dioxide(particularly thiomorpholine). Still further examples include azetidine,piperidone, piperazone, and N-alkyl piperidines such as N-methylpiperidine.

One preferred sub-set of non-aromatic heterocyclic groups consists ofsaturated groups such as azetidine, pyrrolidine, piperidine, morpholine,thiomorpholine, thiomorpholine S,S-dioxide, piperazine, N-alkylpiperazines, and N-alkyl piperidines.

Another sub-set of non-aromatic heterocyclic groups consists ofpyrrolidine, piperidine, morpholine, thiomorpholine, thiomorpholineS,S-dioxide, piperazine and N-alkyl piperazines such as N-methylpiperazine.

One particular sub-set of heterocyclic groups consists of pyrrolidine,piperidine, morpholine and N-alkyl piperazines (e.g. N-methylpiperazine), and optionally thiomorpholine.

Examples of non-aromatic carbocyclic groups include cycloalkane groupssuch as cyclohexyl and cyclopentyl, cycloalkenyl groups such ascyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl, as well ascyclohexadienyl, cyclooctatetraene, tetrahydronaphthenyl and decalinyl.

Preferred non-aromatic carbocyclic groups are monocyclic rings and mostpreferably saturated monocyclic rings.

Typical examples are three, four, five and six membered saturatedcarbocyclic rings, e.g. optionally substituted cyclopentyl andcyclohexyl rings.

One sub-set of non-aromatic carboyclic groups includes unsubstituted orsubstituted (by one or more groups R¹⁰) monocyclic groups andparticularly saturated monocyclic groups, e.g. cycloalkyl groups.Examples of such cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl; more typically cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl, particularly cyclohexyl.

Further examples of non-aromatic cyclic groups include bridged ringsystems such as bicycloalkanes and azabicycloalkanes although suchbridged ring systems are generally less preferred. By “bridged ringsystems” is meant ring systems in which two rings share more than twoatoms, see for example Advanced Organic Chemistry, by Jerry March,4^(th) Edition, Wiley Interscience, pages 131-133, 1992. Examples ofbridged ring systems include bicyclo[2.2.1]heptane,aza-bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane,aza-bicyclo[2.2.2]octane, bicyclo[3.2.1]octane andaza-bicyclo[3.2.1]octane. A particular example of a bridged ring systemis the 1-aza-bicyclo[2.2.2]octan-3-yl group.

Where reference is made herein to carbocyclic and heterocyclic groups,the carbocyclic or heterocyclic ring can, unless the context indicatesotherwise, be unsubstituted or substituted by one or more substituentgroups R¹⁰ selected from halogen, hydroxy, trifluoromethyl, cyano,nitro, carboxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, carbocyclicand heterocyclic groups having from 3 to 12 ring members; a groupR^(a)—R^(b) wherein R^(a) is a bond, O, CO, X¹C(X²), C(X²)X¹, X¹C(X²)X¹,S, SO, SO₂, NR^(c), SO₂NR^(c) or NR^(c)SO₂; and R^(b) is selected fromhydrogen, carbocyclic and heterocyclic groups having from 3 to 12 ringmembers, and a C₁₋₈ hydrocarbyl group optionally substituted by one ormore substituents selected from hydroxy, oxo, halogen, cyano, nitro,carboxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, carbocyclic andheterocyclic groups having from 3 to 12 ring members and wherein one ormore carbon atoms of the C₁₋₈ hydrocarbyl group may optionally bereplaced by O, S, SO, SO₂, NR^(c), X¹C(X²), C(X²)X¹ or X¹C(X²)X¹;

-   -   R^(c) is selected from hydrogen and C₁₋₄ hydrocarbyl; and    -   X¹ is O, S or NR^(c) and X² is ═O, ═S or ═NR^(c).

Where the substituent group R¹⁰ comprises or includes a carbocyclic orheterocyclic group, the said carbocyclic or heterocyclic group may beunsubstituted or may itself be substituted with one or more furthersubstituent groups R¹⁰. In one sub-group of compounds of the formula(I), such further substituent groups R¹⁰ may include carbocyclic orheterocyclic groups, which are typically not themselves furthersubstituted. In another sub-group of compounds of the formula (I), thesaid further substituents do not include carbocyclic or heterocyclicgroups but are otherwise selected from the groups listed above in thedefinition of R¹⁰.

The substituents R¹⁰ may be selected such that they contain no more than20 non-hydrogen atoms, for example, no more than 15 non-hydrogen atoms,e.g. no more than 12, or 11, or 10, or 9, or 8, or 7, or 6, or 5non-hydrogen atoms.

Where the carbocyclic and heterocyclic groups have a pair ofsubstituents on adjacent ring atoms, the two substituents may be linkedso as to form a cyclic group. Thus, two adjacent groups R¹⁰, togetherwith the carbon atoms or heteroatoms to which they are attached may forma 5-membered heteroaryl ring or a 5- or 6-membered non-aromaticcarbocyclic or heterocyclic ring, wherein the said heteroaryl andheterocyclic groups contain up to 3 heteroatom ring members selectedfrom N, O and S. For example, an adjacent pair of substituents onadjacent carbon atoms of a ring may be linked via one or moreheteroatoms and optionally substituted alkylene groups to form a fusedoxa-, dioxa-, aza-, diaza- or oxa-aza-cycloalkyl group.

Examples of such linked substituent groups include:

Examples of halogen substituents include fluorine, chlorine, bromine andiodine. Fluorine and chlorine are particularly preferred.

In the definition of the compounds of the formula (I) above and as usedhereinafter, the term “hydrocarbyl” is a generic term encompassingaliphatic, alicyclic and aromatic groups having an all-carbon backboneand consisting of carbon and hydrogen atoms, except where otherwisestated.

In certain cases, as defined herein, one or more of the carbon atomsmaking up the carbon backbone may be replaced by a specified atom orgroup of atoms.

Examples of hydrocarbyl groups include alkyl, cycloalkyl, cycloalkenyl,carbocyclic aryl, alkenyl, alkynyl, cycloalkylalkyl, cycloalkenylalkyl,and carbocyclic aralkyl, aralkenyl and aralkynyl groups. Such groups canbe unsubstituted or, where stated, substituted by one or moresubstituents as defined herein. The examples and preferences expressedbelow apply to each of the hydrocarbyl substituent groups orhydrocarbyl-containing substituent groups referred to in the variousdefinitions of substituents for compounds of the formula (I) unless thecontext indicates otherwise.

Preferred non-aromatic hydrocarbyl groups are saturated groups such asalkyl and cycloalkyl groups.

Generally by way of example, the hydrocarbyl groups can have up to eightcarbon atoms, unless the context requires otherwise. Within the sub-setof hydrocarbyl groups having 1 to 8 carbon atoms, particular examplesare C₁₋₆ hydrocarbyl groups, such as C₁₋₄ hydrocarbyl groups (e.g. C₁₋₃hydrocarbyl groups or C₁₋₂ hydrocarbyl groups), specific examples beingany individual value or combination of values selected from C₁, C₂, C₃,C₄, C₅, C₆, C₇ and C₈ hydrocarbyl groups.

The term “alkyl” covers both straight chain and branched chain alkylgroups. Examples of alkyl groups include methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl,2-methyl butyl, 3-methyl butyl, and n-hexyl and its isomers. Within thesub-set of alkyl groups having 1 to 8 carbon atoms, particular examplesare C₁₋₆ alkyl groups, such as C₁₋₄ alkyl groups (e.g. C₁₋₃ alkyl groupsor C₁₋₂ alkyl groups).

Examples of cycloalkyl groups are those derived from cyclopropane,cyclobutane, cyclopentane, cyclohexane and cycloheptane. Within thesub-set of cycloalkyl groups the cycloalkyl group will have from 3 to 8carbon atoms, particular examples being C₃₋₆ cycloalkyl groups.

Examples of alkenyl groups include, but are not limited to, ethenyl(vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, butenyl,buta-1,4-dienyl, pentenyl, and hexenyl. Within the sub-set of alkenylgroups the alkenyl group will have 2 to 8 carbon atoms, particularexamples being C₂₋₆ alkenyl groups, such as C₂₋₄ alkenyl groups.

Examples of cycloalkenyl groups include, but are not limited to,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl andcyclohexenyl. Within the sub-set of cycloalkenyl groups the cycloalkenylgroups have from 3 to 8 carbon atoms, and particular examples are C₃₋₆cycloalkenyl groups.

Examples of alkynyl groups include, but are not limited to, ethynyl and2-propynyl (propargyl) groups. Within the sub-set of alkynyl groupshaving 2 to 8 carbon atoms, particular examples are C₂₋₆ alkynyl groups,such as C₂₋₄ alkynyl groups.

Examples of carbocyclic aryl groups include substituted andunsubstituted phenyl groups.

Examples of cycloalkylalkyl, cycloalkenylalkyl, carbocyclic aralkyl,aralkenyl and aralkynyl groups include phenethyl, benzyl, styryl,phenylethynyl, cyclohexylmethyl, cyclopentylmethyl, cyclobutylmethyl,cyclopropylmethyl and cyclopentenylmethyl groups.

When present, and where stated, a hydrocarbyl group can be optionallysubstituted by one or more substituents selected from hydroxy, oxo,alkoxy, carboxy, halogen, cyano, nitro, amino, mono- or di-C₁₋₄hydrocarbylamino, and monocyclic or bicyclic carbocyclic andheterocyclic groups having from 3 to 12 (typically 3 to 10 and moreusually 5 to 10) ring members. Preferred substituents include halogensuch as fluorine. Thus, for example, the substituted hydrocarbyl groupcan be a partially fluorinated or perfluorinated group such asdifluoromethyl or trifluoromethyl. In one embodiment preferredsubstituents include monocyclic carbocyclic and heterocyclic groupshaving 3-7 ring members, more usually 3, 4, 5 or 6 ring members.

Where stated, one or more carbon atoms of a hydrocarbyl group mayoptionally be replaced by O, S, SO, SO₂, NR^(c), X¹C(X²), C(X²)X¹ orX¹C(X²)X¹ (or a sub-group thereof) wherein X¹ and X² are as hereinbeforedefined, provided that at least one carbon atom of the hydrocarbyl groupremains. For example, 1, 2, 3 or 4 carbon atoms of the hydrocarbyl groupmay be replaced by one of the atoms or groups listed, and the replacingatoms or groups may be the same or different. In general, the number oflinear or backbone carbon atoms replaced will correspond to the numberof linear or backbone atoms in the group replacing them. Examples ofgroups in which one or more carbon atom of the hydrocarbyl group havebeen replaced by a replacement atom or group as defined above includeethers and thioethers (C replaced by O or S), amides, esters, thioamidesand thioesters (C—C replaced by X¹C(X²) or C(X²)X¹), sulphones andsulphoxides (C replaced by SO or SO₂), amines (C replaced by NR^(c) ).Further examples include ureas, carbonates and carbamates (C—C—Creplaced by X¹C(X²)X¹).

Where an amino group has two hydrocarbyl substituents, they may,together with the nitrogen atom to which they are attached, andoptionally with another heteroatom such as nitrogen, sulphur, or oxygen,link to form a ring structure of 4 to 7 ring members, more usually 5 to6 ring members.

The term “aza-cycloalkyl” as used herein refers to a cycloalkyl group inwhich one of the carbon ring members has been replaced by a nitrogenatom. Thus examples of aza-cycloalkyl groups include piperidine andpyrrolidine. The term “oxa-cycloalkyl” as used herein refers to acycloalkyl group in which one of the carbon ring members has beenreplaced by an oxygen atom. Thus examples of oxa-cycloalkyl groupsinclude tetrahydrofuran and tetrahydropyran. In an analogous manner, theterms “diaza-cycloalkyl”, “dioxa-cycloalkyl” and “aza-oxa-cycloalkyl”refer respectively to cycloalkyl groups in which two carbon ring membershave been replaced by two nitrogen atoms, or by two oxygen atoms, or byone nitrogen atom and one oxygen atom.

The definition “R^(a)—R^(b)” as used herein, either with regard tosubstituents present on a carbocyclic or heterocyclic moiety, or withregard to other substituents present at other locations on the compoundsof the formula (I), includes inter alia compounds wherein R^(a) isselected from a bond, O, CO, OC(O), SC(O), NR^(c)C(O), OC(S), SC(S),NR^(c)C(S), OC(NR^(c)), SC(NR^(c)), NR^(c)C(NR^(c)), C(O)O, C(O)S,C(O)NR^(c), C(S)O, C(S)S, C(S)NR^(c), C(NR^(c))O, C(NR^(c))S,C(NR^(c))NR^(c), OC(O)O, SC(O)O, NR^(c)C(O)O, OC(S)O, SC(S)O,NR^(c)C(S)O, OC(NR^(c))O, SC(NR^(c))O, NR^(c)C(NR^(c))O, OC(O)S, SC(O)S,NR^(c)C(O)S, OC(S)S, SC(S)S, NR^(c)C(S)S, OC(NR^(c))S, SC(NR^(c))S,NR^(c)C(NR^(c))S, OC(O)NR^(c), SC(O)NR^(c), NR^(c)C(O) NR^(c),OC(S)NR^(c), SC(S) NR^(c), NR^(c)C(S)NR^(c), OC(NR^(c))NR^(c),SC(NR^(c))NR^(c), NR^(c)C(NR^(c)NR^(c), S, SO, SO₂NR^(c), SO₂NR^(c) andNR^(c)SO₂ wherein R^(c) is as hereinbefore defined.

The moiety R^(b) can be hydrogen or it can be a group selected fromcarbocyclic and heterocyclic groups having from 3 to 12 ring members(typically 3 to 10 and more usually from 5 to 10), and a C₁₋₈hydrocarbyl group optionally substituted as hereinbefore defined.Examples of hydrocarbyl, carbocyclic and heterocyclic groups are as setout above.

When R^(a) is O and R^(b) is a C₁₋₈ hydrocarbyl group, R^(a) and R^(b)together form a hydrocarbyloxy group. Preferred hydrocarbyloxy groupsinclude saturated hydrocarbyloxy such as alkoxy (e.g. C₁₋₆ alkoxy, moreusually C₁₋₄ alkoxy such as ethoxy and methoxy, particularly methoxy),cycloalkoxy (e.g. C₃₋₆ cycloalkoxy such as cyclopropyloxy,cyclobutyloxy, cyclopentyloxy and cyclohexyloxy) and cycloalkyalkoxy(e.g. C₃₋₆ cycloalkyl-C₁₋₂ alkoxy such as cyclopropylmethoxy).

The hydrocarbyloxy groups can be substituted by various substituents asdefined herein. For example, the alkoxy groups can be substituted byhalogen (e.g. as in difluoromethoxy and trifluoromethoxy), hydroxy (e.g.as in hydroxyethoxy), C₁₋₂ alkoxy (e.g. as in methoxyethoxy),hydroxy-C₁₋₂ alkyl (as in hydroxyethoxyethoxy) or a cyclic group (e.g. acycloalkyl group or non-aromatic heterocyclic group as hereinbeforedefined). Examples of alkoxy groups bearing a non-aromatic heterocyclicgroup as a substituent are those in which the heterocyclic group is asaturated cyclic amine such as morpholine, piperidine, pyrrolidine,piperazine, C₁₋₄-alkyl-piperazines, C₃₋₇-cycloalkyl-piperazines,tetrahydropyran or tetrahydrofuran and the alkoxy group is a C₁₋₄ alkoxygroup, more typically a C₁₋₃ alkoxy group such as methoxy, ethoxy orn-propoxy.

Alkoxy groups substituted by a monocyclic group such as pyrrolidine,piperidine, morpholine and piperazine and N-substituted derivativesthereof such as N-benzyl, N-C₁₋₄ acyl and N-C₁₋₄ alkoxycarbonyl.Particular examples include pyrrolidinoethoxy, piperidinoethoxy andpiperazinoethoxy.

When R^(a) is a bond and R^(b) is a C₁₋₈ hydrocarbyl group, examples ofhydrocarbyl groups R^(a)—R^(b) are as hereinbefore defined. Thehydrocarbyl groups may be saturated groups such as cycloalkyl and alkyland particular examples of such groups include methyl, ethyl andcyclopropyl. The hydrocarbyl (e.g. alkyl) groups can be substituted byvarious groups and atoms as defined herein. Examples of substitutedalkyl groups include alkyl groups substituted by one or more halogenatoms such as fluorine and chlorine (particular examples includingbromoethyl, chloroethyl and trifluoromethyl), or hydroxy (e.g.hydroxymethyl and hydroxyethyl), C₁₋₈ acyloxy (e.g. acetoxymethyl andbenzyloxymethyl), amino and mono- and dialkylamino (e.g. aminoethyl,methylaminoethyl, dimethylaminomethyl, dimethylaminoethyl andtert-butylaminomethyl), alkoxy (e.g. C₁₋₂ alkoxy such as methoxy—as inmethoxyethyl), and cyclic groups such as cycloalkyl groups, aryl groups,heteroaryl groups and non-aromatic heterocyclic groups as hereinbeforedefined).

Particular examples of alkyl groups substituted by a cyclic group arethose wherein the cyclic group is a saturated cyclic amine such asmorpholine, piperidine, pyrrolidine, piperazine, C₁₋₄-alkyl-piperazines,C₃₋₇-cycloalkyl-piperazines, tetrahydropyran or tetrahydrofuran and thealkyl group is a C₁₋₄ alkyl group, more typically a C₁₋₃ alkyl groupsuch as methyl, ethyl or n-propyl. Specific examples of alkyl groupssubstituted by a cyclic group include pyrrolidinomethyl,pyrrolidinopropyl, morpholinomethyl, morpholinoethyl, morpholinopropyl,piperidinylmethyl, piperazinomethyl and N-substituted forms thereof asdefined herein.

Particular examples of alkyl groups substituted by aryl groups andheteroaryl groups include benzyl and pyridylmethyl groups.

When R^(a) is SO₂NR^(c), R^(b) can be, for example, hydrogen or anoptionally substituted C₁₋₈ hydrocarbyl group, or a carbocyclic orheterocyclic group. Examples of R^(a)—R^(b) where R^(a) is SO₂NR^(c)include aminosulphonyl, C₁₋₄ alkylaminosulphonyl and di-C₁₋₄alkylaminosulphonyl groups, and sulphonamides formed from a cyclic aminogroup such as piperidine, morpholine, pyrrolidine, or an optionallyN-substituted piperazine such as N-methyl piperazine.

Examples of groups R^(a)—R^(b) where R^(a) is SO₂ includealkylsulphonyl, heteroarylsulphonyl and arylsulphonyl groups,particularly monocyclic aryl and heteroaryl sulphonyl groups. Particularexamples include methylsulphonyl, phenylsulphonyl and toluenesulphonyl.

When R^(a) is NR^(c), R^(b) can be, for example, hydrogen or anoptionally substituted C₁₋₈ hydrocarbyl group, or a carbocyclic orheterocyclic group. Examples of R^(a)—R^(b) where R^(a) is NR^(c)include amino, C₁₋₄ alkylamino (e.g. methylamino, ethylamino,propylamino, isopropylamino, tert-butylamino), di-C₁₋₄ alkylamino (e.g.dimethylamino and diethylamino) and cycloalkylamino (e.g.cyclopropylamino, cyclopentylamino and cyclohexylamino).

Specific Embodiments of and Preferences for Moieties X, Y, A, R^(g), R¹to R⁴ and R¹⁰

X

In formula (I), X is a group R¹-A-NR⁴— or a 5- or 6-membered carbocyclicor heterocyclic ring.

In one embodiment, X is a group R¹-A-NR⁴—.

In another embodiment, X is a 5- or 6-membered carbocyclic orheterocyclic ring.

A

In formula (I), A is a bond, C═O, NR^(g)(C═O) or O(C═O). It will beappreciated that the moiety R¹-A-NR⁴ linked to the 4-position of thepyrazole ring can therefore take the form of an amine R¹—NR⁴, an amideR¹—C(═O)NR⁴, a urea R¹—NR^(g)C(═O)NR⁴ or a carbamate R¹—OC(═O)NR⁴.

In one preferred group of compounds of the invention, A is C═O and hencethe group R¹-A-NR⁴ takes the form of an amide R¹—C(═O)NR⁴. In anothergroup of compounds of the invention, A is a bond and hence the groupR¹-A-NR⁴ takes the form of an amine R¹—NR⁴.

R⁴

R⁴ is hydrogen or a C₁₋₄ hydrocarbyl group optionally substituted byhalogen (e.g. fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy).

The number of optional subsitutents on the hydrocarbyl group typicallywill vary according to the nature of the substituent. For example, wherethe substituent is halogen, there may be from one to three halogen atomspresent, preferably two or three. Where the substituent is hydroxyl oran alkoxy group, typically there will be only a single such substituentpresent

R⁴ is preferably hydrogen or C₁₋₃ alkyl, more preferably hydrogen ormethyl and most preferably is hydrogen.

R^(g)

R^(g) is hydrogen or a C₁₋₄ hydrocarbyl group optionally substituted byhydroxyl or C₁₋₄ alkoxy (e.g. methoxy).

When R^(g) is C₁₋₄ hydrocarbyl substituted by hydroxyl or C₁₋₄ alkoxy,typically there is only one such substituent present.

Preferably R^(g) is hydrogen or C₁₋₃ alkyl, more preferably hydrogen ormethyl and most preferably R^(g) is hydrogen.

R²

R² is hydrogen, halogen, C₁₋₄ alkoxy, or a C₁₋₄ hydrocarbyl groupoptionally substituted by halogen, hydroxyl or C₁₋₄ alkoxy.

When R² is halogen, preferably it is selected from chlorine and fluorineand more preferably it is fluorine.

When R² is C₁₋₄ alkoxy, it can be, for example, C₁₋₃ alkoxy, morepreferably C₁₋₂ alkoxy and most preferably methoxy.

When R² is an optionally substituted C₁₋₄ hydrocarbyl group, thehydrocarbyl group is preferably a C₁₋₃ hydrocarbyl group, morepreferably a C₁₋₂ hydrocarbyl group, for example an optionallysubstituted methyl group. The optional substituents for the optionallysubstituted hydrocarbyl group are preferably selected from fluorine,hydroxyl and methoxy.

The number of optional substituents on the hydrocarbyl group typicallywill vary according to the nature of the substituent. For example, wherethe substituent is halogen, there may be from one to three halogen atomspresent, preferably two or three. Where the substituent is hydroxyl ormethoxy, typically there will be only a single such substituent present.

The hydrocarbyl groups constituting R² are preferably saturatedhydrocarbyl groups. Examples of saturated hydrocarbyl groups includemethyl, ethyl, n-propyl, i-propyl and cyclopropyl.

In one embodiment, R² is hydrogen, halogen, C₁₋₄ alkoxy, or a C₁₋₄hydrocarbyl group optionally substituted by halogen, hydroxyl or C₁₋₄alkoxy.

In another embodiment, R² is hydrogen, fluorine, chlorine, methoxy, or aC₁₋₃ hydrocarbyl group optionally substituted by fluorine, hydroxyl ormethoxy.

In a preferred embodiment, R² is hydrogen or methyl, most preferablyhydrogen.

R¹

R¹ is hydrogen, a carbocyclic or heterocyclic group having from 3 to 12ring members, or a C₁₋₈ hydrocarbyl group optionally substituted by oneor more substituents selected from halogen (e.g. fluorine), hydroxy,C₁₋₄ hydrocarbyloxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, andcarbocyclic or heterocyclic groups having from 3 to 12 ring members, andwherein 1 or 2 of the carbon atoms of the hydrocarbyl group mayoptionally be replaced by an atom or group selected from O, S, NH, SO,SO₂. Examples of carbocyclic or heterocyclic groups and hydrocarbylgroups and general preferences for such groups are as set out above inthe General Preferences and Definitions section, and as set out below.

In one embodiment, R¹ is an aryl or heteroaryl group.

When R¹ is a heteroaryl group, particular heteroaryl groups includemonocyclic heteroaryl groups containing up to three heteroatom ringmembers selected from O, S and N, and bicyclic heteroaryl groupscontaining up to 2 heteroatom ring members selected from O, S and N andwherein both rings are aromatic.

Examples of such groups include furanyl (e.g. 2-furanyl or 3-furanyl),indolyl (e.g. 3-indolyl, 6-indolyl), 2,3-dihydro-benzo[1,4]dioxinyl(e.g. 2,3-dihydro-benzo[1,4]dioxin-5-yl), pyrazolyl (e.g.pyrazole-5-yl), pyrazolo[1,5-a]pyridinyl (e.g.pyrazolo[1,5-a]pyridine-3-yl), oxazolyl (e.g.), isoxazolyl (e.g.isoxazol-4-yl), pyridyl (e.g. 2-pyridyl, 3-pyridyl, 4-pyridyl),quinolinyl (e.g. 2-quinolinyl), pyrrolyl (e.g. 3-pyrrolyl), imidazolyland thienyl (e.g. 2-thienyl, 3-thienyl).

One sub-group of heteroaryl groups R¹ consists of furanyl (e.g.2-furanyl or 3-furanyl), indolyl, oxazolyl, isoxazolyl, pyridyl,quinolinyl, pyrrolyl, imidazolyl and thienyl.

A preferred sub-set of R¹ heteroaryl groups includes 2-furanyl,3-furanyl, pyrrolyl, imidazolyl and thienyl.

Preferred aryl groups R¹ are phenyl groups.

The group R¹ can be an unsubstituted or substituted carbocylic orheterocyclic group in which one or more substituents can be selectedfrom the group R¹⁰ as hereinbefore defined. In one embodiment, thesubstituents on R¹ may be selected from the group R^(10a) consisting ofhalogen, hydroxy, trifluoromethyl, cyano, nitro, carboxy, a groupR^(a)—R^(b) wherein R^(a) is a bond, O, CO, X³C(X⁴), C(X⁴)X³, X³C(X⁴)X³,S, SO, SO₂, and R^(b) is selected from hydrogen and a C₁₋₈ hydrocarbylgroup optionally substituted by one or more substituents selected fromhydroxy, oxo, halogen, cyano, nitro, carboxy and monocyclic non-aromaticcarbocyclic or heterocyclic groups having from 3 to 6 ring members;wherein one or more carbon atoms of the C₁₋₈ hydrocarbyl group mayoptionally be replaced by O, S, SO, SO₂, X³C(X⁴), C(X⁴)X³ or X³C(X⁴)X³;X³ is O or S; and X⁴ is ═O or ═S.

Where the carbocyclic and heterocyclic groups have a pair ofsubstituents on adjacent ring atoms, the two substituents may be linkedso as to form a cyclic group. Thus, two adjacent groups R¹⁰, togetherwith the carbon atoms or heteroatoms to which they are attached may forma 5-membered heteroaryl ring or a 5- or 6-membered non-aromaticcarbocyclic or heterocyclic ring, wherein the said heteroaryl andheterocyclic groups contain up to 3 heteroatom ring members selectedfrom N, O and S. In particular the two adjacent groups R¹⁰, togetherwith the carbon atoms or heteroatoms to which they are attached, mayform a 6-membered non-aromatic heterocyclic ring, containing up to 3, inparticular 2, heteroatom ring members selected from N, O and S. Moreparticularly the two adjacent groups R¹⁰ may form a 6-memberednon-aromatic heterocyclic ring, containing 2 heteroatom ring membersselected from N, or O, such as dioxan e.g. [1,4 dioxan]. In oneembodiment R¹ is a carbocyclic group e.g. phenyl having a pair ofsubstituents on adjacent ring atoms linked so as to form a cyclic groupe.g. to form 2,3-dihydro-benzo[1,4]dioxine.

More particularly, the substituents on R¹ may be selected from halogen,hydroxy, trifluoromethyl, a group R^(a)—R^(b) wherein R^(a) is a bond orO, and R^(b) is selected from hydrogen and a C₁₋₄ hydrocarbyl groupoptionally substituted by one or more substituents selected fromhydroxyl, halogen (preferably fluorine) and 5 and 6 membered saturatedcarbocyclic and heterocyclic groups (for example groups containing up totwo heteroatoms selected from O, S and N, such as unsubstitutedpiperidine, pyrrolidino, morpholino, piperazino and N-methylpiperazino).

The group R¹ may be substituted by more than one substituent. Thus, forexample, there may be 1 or 2 or 3 or 4 substituents. In one embodiment,where R¹ is a six membered ring (e.g. a carbocyclic ring such as aphenyl ring), there may be one, two or three substituents and these maybe located at the 2-, 3-, 4- or 6-positions around the ring. By way ofexample, a phenyl group R¹ may be 2-monosubstituted, 3-monosubstituted,2,6-disubstituted, 2,3-disubstituted, 2,4-disubstituted2,5-disubstituted, 2,3,6-trisubstituted or 2,4,6-trisubstituted. Moreparticularly, a phenyl group R¹ may be monosubstituted at the 2-positionor disubstituted at positions 2- and 6- with substituents selected fromfluorine, chlorine and R^(a)—R^(b), where R^(a) is O and R^(b) is C₁₋₄alkyl (e.g. methyl or ethyl). In one embodiment, fluorine is a preferredsubstituent. In another embodiment, preferred substituents are selectedfrom fluorine, chlorine and methoxy.

Particular examples of non-aromatic groups R¹ include unsubstituted orsubstituted (by one or more groups R¹⁰) monocyclic cycloalkyl groups.Examples of such cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl; more typically cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl, particularly cyclohexyl.

Further examples of non-aromatic groups R¹ include unsubstituted orsubstituted (by one or more groups R¹⁰) heterocyclic groups having from3 to 12 ring members, typically 4 to 12 ring members, and more usuallyfrom 5 to 10 ring members. Such groups can be monocyclic or bicyclic,for example, and typically have from 1 to 5 heteroatom ring members(more usually 1, 2, 3 or 4 heteroatom ring members) typically selectedfrom nitrogen, oxygen and sulphur.

When sulphur is present, it may, where the nature of the adjacent atomsand groups permits, exist as —S—, —S(O)— or —S(O)₂—.

The heterocylic groups can contain, for example, cyclic ether moieties(e.g as in tetrahydrofuran and dioxane), cyclic thioether moieties (e.g.as in tetrahydrothiophene and dithiane), cyclic amine moieties (e.g. asin pyrrolidine), cyclic amides (e.g. as in pyrrolidone), cyclic esters(e.g. as in butyrolactone), cyclic thioamides and thioesters, cyclicsulphones (e.g. as in sulpholane and sulpholene), cyclic sulphoxides,cyclic sulphonamides and combinations thereof (e.g. morpholine andthiomorpholine and its S-oxide and S,S-dioxide).

In one sub-set of heterocyclic groups R¹, the heterocyclic groupscontain cyclic ether moieties (e.g as in tetrahydrofuran and dioxane),cyclic thioether moieties (e.g. as in tetrahydrothiophene and dithiane),cyclic amine moieties (e.g. as in pyrrolidine), cyclic sulphones (e.g.as in sulpholane and sulpholene), cyclic sulphoxides, cyclicsulphonamides and combinations thereof (e.g. thiomorpholine).

Examples of monocyclic non-aromatic heterocyclic groups R¹ include 5-,6-and 7-membered monocyclic heterocyclic groups such as morpholine,piperidine (e.g. 1-piperidinyl, 2-piperidinyl 3-piperidinyl and4-piperidinyl), pyrrolidine (e.g. 1-pyrrolidinyl, 2-pyrrolidinyl and3-pyrrolidinyl), pyrrolidone, pyran (2H-pyran or 4H-pyran),dihydrothiophene, dihydropyran, dihydrofuran, dihydrothiazole,tetrahydrofuran, tetrahydrothiophene, dioxane, tetrahydropyran (e.g.4-tetrahydro pyranyl), imidazoline, imidazolidinone, oxazoline,thiazoline, 2-pyrazoline, pyrazolidine, piperazine, and N-alkylpiperazines such as N-methyl piperazine. Further examples includethiomorpholine and its S-oxide and S,S-dioxide (particularlythiomorpholine). Still further examples include N-alkyl piperidines suchas N-methyl piperidine.

One sub-group of non-aromatic heterocyclic groups R¹ includesunsubstituted or substituted (by one or more groups R¹⁰) 5-, 6-and7-membered monocyclic heterocyclic groups such as morpholine, piperidine(e.g. 1-piperidinyl, 2-piperidinyl 3-piperidinyl and 4-piperidinyl),pyrrolidine (e.g. 1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl),pyrrolidone, piperazine, and N-alkyl piperazines such as N-methylpiperazine, wherein a particular sub-set consists of pyrrolidine,piperidine, morpholine, thiomorpholine and N-methyl piperazine.

In general, preferred non-aromatic heterocyclic groups includepyrrolidine, piperidine, morpholine, thiomorpholine, thiomorpholineS,S-dioxide, piperazine, N-alkyl piperazines, and N-alkyl piperidines.

Another particular sub-set of heterocyclic groups consists ofpyrrolidine, piperidine, morpholine and N-alkyl piperazines, andoptionally, N-methyl piperazine and thiomorpholine.

When R¹ is a C₁₋₈ hydrocarbyl group substituted by a carbocyclic orheterocyclic group, the carbocyclic and heterocyclic groups can bearomatic or non-aromatic and can be selected from the examples of suchgroups set out hereinabove. The substituted hydrocarbyl group istypically a saturated C₁₋₄ hydrocarbyl group such as an alkyl group,preferably a CH₂ or CH₂CH₂ group. Where the substituted hydrocarbylgroup is a C₂₋₄ hydrocarbyl group, one of the carbon atoms and itsassociated hydrogen atoms may be replaced by a sulphonyl group, forexample as in the moiety SO₂CH₂.

When the carbocyclic or heterocylic group attached to the a C₁₋₈hydrocarbyl group is aromatic, examples of such groups includemonocyclic aryl groups and monocyclic heteroaryl groups containing up tofour heteroatom ring members selected from O, S and N, and bicyclicheteroaryl groups containing up to 2 heteroatom ring members selectedfrom O, S and N and wherein both rings are aromatic.

Examples of such groups are set out in the “General Preferences andDefinitions” section above.

Particular examples of such groups include furanyl (e.g. 2-furanyl or3-furanyl), indolyl, oxazolyl, isoxazolyl, pyridyl, quinolinyl,pyrrolyl, imidazolyl and thienyl. Particular examples of aryl andheteroaryl groups as substituents for a C₁₋₈ hydrocarbyl group includephenyl, imidazolyl, tetrazolyl, triazolyl, indolyl, 2-furanyl,3-furanyl, pyrrolyl and thienyl. Such groups may be substituted by oneor more substituents R¹⁰ or R^(10a) as defined herein.

When R¹ is a C₁₋₈ hydrocarbyl group substituted by a non-aromaticcarbocyclic or heterocyclic group, the non-aromatic or heterocyclicgroup may be a group selected from the lists of such groups set outhereinabove. For example, the non-aromatic group can be a monocyclicgroup having from 4 to 7 ring members, e.g. 5 to 7 ring members, andtypically containing from 0 to 3, more typically 0, 1 or 2, heteroatomring members selected from O, S and N. When the cyclic group is acarbocyclic group, it may additionally be selected from monocyclicgroups having 3 ring members. Particular examples include monocycliccycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl, and 5-, 6-and 7-membered monocyclicheterocyclic groups such as morpholine, piperidine (e.g. 1-piperidinyl,2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g.1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone,piperazine, and N-alkyl piperazines such as N-methyl piperazine. Ingeneral, preferred non-aromatic heterocyclic groups include pyrrolidine,piperidine, morpholine, thiomorpholine and N-methyl piperazine.

When R¹ is an optionally substituted C₁₋₈ hydrocarbyl group, thehydrocarbyl group may be as hereinbefore defined, and is preferably upto four carbon atoms in length, more usually up to three carbon atoms inlength for example one or two carbon atoms in length.

In one embodiment, the hydrocarbyl group is saturated and may be acyclicor cyclic, for example acyclic. An acyclic saturated hydrocarbyl group(i.e. an alkyl group) may be a straight chain or branched alkyl group.

Examples of straight chain alkyl groups R¹ include methyl, ethyl, propyland butyl.

Examples of branched chain alkyl groups R¹ include isopropyl, isobutyl,tent-butyl and 2,2-dimethylpropyl.

In one embodiment, the hydrocarbyl group is a linear saturated grouphaving from 1-6 carbon atoms, more usually 1-4 carbon atoms, for example1-3 carbon atoms, e.g. 1, 2 or 3 carbon atoms. When the hydrocarbylgroup is substituted, particular examples of such groups are substituted(e.g. by a carbocyclic or heterocyclic group) methyl and ethyl groups.

A C₁₋₈ hydrocarbyl group R¹ can be optionally substituted by one or moresubstituents selected from halogen (e.g. fluorine), hydroxy, C₁₋₄hydrocarbyloxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, andcarbocyclic or heterocyclic groups having from 3 to 12 ring members, andwherein 1 or 2 of the carbon atoms of the hydrocarbyl group mayoptionally be replaced by an atom or group selected from O, S, NH, SO,SO₂. Particular substituents for the hydrocarbyl group include hydroxy,chlorine, fluorine (e.g. as in trifluoromethyl), methoxy, ethoxy, amino,methylamino and dimethylamino, preferred substituents being hydroxy andfluorine.

When A is C═O, particular groups R¹—CO are the groups set out in Table 1below.

In Table 1, the point of attachment of the group to the nitrogen atom ofthe pyrazole-4-amino group is represented by the terminal single bondextending from the carbonyl group. Thus, by way of illustration, group Bin the table is the trifluoroacetyl group, group D in the table is thephenylacetyl group and group I in the table is the3-(4-chlorophenyl)propionyl group.

TABLE 1 Examples of the group R¹—CO CH₃—C(═O)— A CF₃—C(═O)— B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

AB

AC

AD

AE

AF

AG

AH

AI

AJ

AK

AL

AM

AN

AO

AP

AQ

AR

AS

AT

AU

AV

AW

AX

AY

AZ

BA

BB

BC

BD

BE

BF

BG

BH

BI

BJ

BK

BL

BM

BN

BO

BP

BQ

BR

BS

BT

BU

BV

BW

BX

BY

BZ

BAA

BAB

BAC

BAD

BAE

BAF

BAG

BAH

BAI

BAJ

BAK

BAL

BAM

BAN

BAO

One sub-group of groups R¹—CO consists of groups A to BF in Table 1above.

Another sub-group of groups R¹—CO consists of groups A to BS in Table 1above.

One set of preferred groups R¹—CO consists of the groups J, AB, AH, AJ,AL, AS, AX, AY, AZ, BA, BB, BD, BH, BL, BQ, BS and BAI

Another set of preferred groups R¹—CO consists of the groups J, AB, AH,AJ, AL, AS, AX, AY, AZ, BA, BB, BD, BH, BL, BQ and BS.

More preferred groups R¹—CO— are AJ, AX, BQ, BS and BAI.

One particularly preferred sub-set of groups R¹—CO— consists of AJ, BQand BS.

Another particularly preferred sub-set of groups R¹—CO— consists of AJand BQ.

When X is R¹-A-NR⁴ and A is C═O, and R¹ is a phenyl ring bearing asubstituent at the 4-position, the substituent at the 4-position ispreferably other than a phenyl group having a group SO₂NH₂ or SO₂Me atthe ortho-position.

In one general embodiment, R¹ may be other than a substituted orunsubstituted tetrahydroquinoline, chroman, chromene, thiochroman,thiochromene, dihydro-naphthalene or tetrahydronaphthalene group. Moreparticularly, R¹ may be other than a substituted or unsubstitutedtetrahydroquinoline, chroman, chromene, thiochroman, thiochromene,dihydro-naphthalene or tetrahydronaphthalene group linked by itsaromatic ring to the moiety A-NR⁴—.

In another general embodiment, when R¹ is a substituted or unsubstitutedphenyl group, the moiety Y—R³ may be other than hydrogen, unsubstitutedC₁₋₁₀ alkyl, unsubstituted C₅₋₁₀ cycloalkyl, unsubstituted phenyl,unsubstituted C₁₋₁₀ alkylphenyl or unsubstituted phenyl-C₁₋₁₀ alkyl.

In the context of the group R¹-A-NR⁴—, when R¹ is an optionallysubstituted hydrocarbyl group and the hydrocarbyl group comprises orcontains a substituted or unsubstituted alkene group, it is preferredthat the carbon-carbon double bond of the alkene group is not directlybonded to the group A.

Also in the context of the group R¹-A-NR⁴—, when R¹ is an optionallysubstituted hydrocarbyl group, the hydrocarbyl group may be other thanan alkene group.

In another general embodiment, when Y is a bond, R³ is hydrogen, A is COand R¹ is a substituted phenyl group, each substituent on the phenylgroup may be other than a group CH₂—P(O)R^(x)R^(y) where R^(x) and R^(y)are each selected from alkoxy and phenyl groups.

Y

In the compounds of the formula (I), Y is a bond or an alkylene chain of1, 2 or 3 carbon atoms in length.

The term “alkylene” has its usual meaning and refers to a divalentsaturated acyclic hydrocarbon chain. The hydrocarbon chain may bebranched or unbranched. Where an alkylene chain is branched, it may haveone or more methyl group side chains. Examples of alkylene groupsinclude —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, CH(CH₃)—, —C(CH₃)₂—,—CH₂—CH(CH₃)—, —CH₂—C(CH₃)₂— and —CH(CH₃)—CH(CH₃)—.

In one embodiment, Y is a bond.

In another embodiment, Y is an alkylene chain.

When Y is an alkylene chain, preferably it is unbranched and moreparticularly contains 1 or 2 carbon atoms, preferably 1 carbon atom.Thus preferred groups Y are —CH₂— and —CH₂—CH₂—, a most preferred groupbeing (CH₂)—.

Where Y is a branched chain, preferably it has no more than two methylside chains. For example, it may have a single methyl side chain. In oneembodiment, Y is a group —CH(Me)-.

In one sub-group of compounds, Y is a bond, CH₂, CH₂CH₂ or CH₂CH(CH₃).

R³

The group R³ is selected from hydrogen and carbocyclic and heterocyclicgroups having from 3 to 12 ring members.

In one sub-group of compounds, Y is a bond and R³ is hydrogen.

In another sub-group of compounds Y is an alkylene chain as hereinbeforedefined and R³ is hydrogen.

In a another sub-group of compounds, Y is a bond or an alkylene chain(e.g. a group —(CH₂)—) and R³ is a carbocyclic or heterocyclic group.

In a further sub-group of compounds, Y is a bond and R³ is a carbocyclicor heterocyclic group.

In a still further sub-group of compounds, Y is an alkylene chain (e.g.a group —(CH₂)—) and R³ is a carbocyclic or heterocyclic group.

The carbocyclic and heterocyclic groups R³ can be aryl, heteroaryl,non-aromatic carbocyclic or non-aromatic heterocyclic and examples ofsuch groups are as set out in detail above in the General Preferencesand Definitions section, and as set out below.

Preferred aryl groups R³ are unsubstituted and substituted phenylgroups.

Examples of heteroaryl groups R³ include monocyclic heteroaryl groupscontaining up to three (and more preferably up to two) heteroatom ringmembers selected from O, S and N. Preferred heteroaryl groups includefive membered rings containing one or two heteroatom ring members andsix membered rings containing a single heteroatom ring member, mostpreferably nitrogen. Particular examples of heteroaryl groups includeunsubstituted or substituted pyridyl, imidazole, pyrazole, thiazole,isothiazole, isoxazole, oxazole, furyl and thiophene groups.

Particular heteroaryl groups are unsubstituted and substituted pyridylgroups, e.g. 2-pyridyl, 3-pyridyl and 4-pyridyl groups, especially 3-and 4-pyridyl groups. When the pyridyl groups are substituted, they canbear one or more substituents, typically no more than two, and moreusually one substituent selected, for example, from C₁₋₄ alkyl (e.g.methyl), halogen (e.g. fluorine or chlorine, preferably chlorine), andC₁₋₄ alkoxy (e.g. methoxy). Substituents on the pyridyl group mayfurther be selected from amino, mono-C₁₋₄ alkylamino and di-C₁₋₄alkylamino, particularly amino.

In one embodiment, when R³ is an aryl (e.g. phenyl) or heteroaryl group,the substituents on the carbocyclic or heterocyclic group may beselected from the group R^(10a) consisting of halogen, hydroxy,trifluoromethyl, cyano, monocyclic carbocyclic and heterocyclic groupshaving from 3 to 7 (typically 5 or 6) ring members, and a groupR^(a)—R^(b) wherein R^(a) is a bond, O, CO, X¹C(X²), C(X²)X¹, X¹C(X²)X¹,S, SO, SO₂, NR^(c), SO₂NR^(c) or NR^(c)SO₂; and R^(b) is selected fromhydrogen, a carbocyclic or heterocyclic group with 3-7 ring members anda C₁₋₈ hydrocarbyl group optionally substituted by one or moresubstituents selected from hydroxy, oxo, halogen, cyano, nitro, carboxy,amino, mono- or di-C₁₋₄ hydrocarbylamino, a carbocyclic or heterocyclicgroup with 3-7 ring members and wherein one or more carbon atoms of theC₁₋₈ hydrocarbyl group may optionally be replaced by O, S, SO,SO₂NR^(c), X¹C(X²), C(X²)X¹ or X¹C(X²)X¹; and R^(c), X¹ and X² are ashereinbefore defined.

Examples of non-aromatic groups R³ include optionally substituted (byR¹⁰ or R^(10a)) cycloalkyl, oxa-cycloalkyl, aza-cycloalkyl,diaza-cycloalkyl, dioxa-cycloalkyl and aza-oxa-cycloalkyl groups.Further examples include C₇₋₁₀ aza-bicycloalkyl groups such as1-aza-bicyclo[2.2.2]octan-3-yl.

Particular examples of such groups include unsubstituted or substitutedcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyran,morpholine, tetrahydrofuran, piperidine and pyrrolidine groups.

One sub-set of non-aromatic groups R³ consists of cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyran, tetrahydrofuran,piperidine and pyrrolidine groups.

Preferred non-aromatic groups R³ include unsubstituted or substitutedcyclopentyl, cyclohexyl, tetrahydropyran, tetrahydrofuran, piperidineand pyrrolidine groups,

The non-aromatic groups may be unsubstituted or substituted with one ormore groups R¹⁰ or R^(10a) as hereinbefore defined.

Particular substituents for R³ (e.g. (i) when R³ is an aryl orheteroaryl group or (ii) when R³ is a non-aromatic group) are selectedfrom the group R^(10a) consisting of halogen; hydroxy; monocycliccarbocyclic and heterocyclic groups having from 3 to 6 ring members andcontaining up to 2 heteroataom ring members selected from O, N and S;and a group R^(a)—R^(b) wherein R^(a) is a bond, O, CO, CO₂, SO₂NH,SO₂NH or NHSO₂; and R^(b) is selected from hydrogen, a carbocyclic orheterocyclic group with 3-6 ring members and containing up to 2heteroatom ring members selected from O, N and S; and a C₁₋₆ hydrocarbylgroup optionally substituted by one or more substituents selected fromhydroxy, oxo, halogen, carboxy, amino, mono- or di-C₁₋₄hydrocarbylamino, a carbocyclic or heterocyclic group with 3-6 ringmembers and containing up to 2 heteroatom ring members selected from O,N and S; and wherein one or two carbon atoms of the C₁₋₆ hydrocarbylgroup may optionally be replaced by O, S, SO, SO₂ or NH.

In one embodiment, preferred R^(10a) substituent groups on R³ (e.g. (i)when R³ is an aryl or heteroaryl group or (ii) when R³ is a non-aromaticgroup) include halogen, a group R^(a)—R^(b) wherein R^(a) is a bond, O,CO, C(X²)X¹, and R^(b) is selected from hydrogen, heterocyclic groupshaving 3-7 ring members and a C₁₋₄ hydrocarbyl group optionallysubstituted by one or more substituents selected from hydroxy, carboxy,amino, mono- or di-C₁₋₄ hydrocarbylamino, and heterocyclic groups having3-7 ring members.

Particularly preferred substituent groups R^(10a) on R³ (e.g. (i) whenR³ is an aryl or heteroaryl group or (ii) when R³ is a non-aromaticgroup) include halogen, especially fluorine, C₁₋₃ alkoxy such asmethoxy, and C₁₋₃ hydrocarbyl optionally substituted by fluorine,hydroxy (e.g. hydroxymethyl), C₁₋₂ alkoxy or a 5- or 6-memberedsaturated heterocyclic ring such as piperidino, morpholino, piperazinoand N-methylpiperazino.

In another embodiment, the substituents for R³ (whether aromatic ornon-aromatic) are selected from:

-   -   halogen (e.g. fluorine and chlorine)    -   C₁₋₄ alkoxy (e.g. methoxy and ethoxy) optionally substituted by        one or substituents selected from halogen, hydroxy, C₁₋₂ alkoxy        and five and six membered saturated heterocyclic rings        containing 1 or 2 heteroatoms selected from O, N and S, the        heterocyclic rings being optionally further substituted by one        or more C₁₋₄ groups (e.g. methyl) and wherein the S, when        present, may be present as S, SO or SO₂;    -   C₁₋₄ alkyl optionally substituted by one or substituents        selected from halogen, hydroxy, C₁₋₄ alkoxy, amino, C₁₋₄        alkylsulphonylamino, 3 to 6 membered cycloalkyl groups (e.g.        cyclopropyl), phenyl (optionally substituted by one or more        substituents selected from halogen, methyl, methoxy and amino)        and five and six membered saturated heterocyclic rings        containing 1 or 2 heteroatoms selected from O, N and S, the        heterocyclic rings being optionally further substituted by one        or more C₁₋₄ groups (e.g. methyl) and wherein the S, when        present, may be present as S, SO or SO₂;    -   hydroxy;    -   amino, mono-C₁₋₄ alkylamino, di-C₁₋₄ alkylamino,        benzyloxycarbonylamino and C₁₋₄ alkoxycarbonylamino;    -   carboxy and C₁₋₄ alkoxycarbonyl;    -   C₁₋₄ alkylaminosulphonyl and C₁₋₄ alkylsulphonylamino;    -   C₁₋₄ alkylsulphonyl;    -   a group O-Het^(s) or NH-Het^(s) where Het^(s) is a five or six        membered saturated heterocyclic ring containing 1 or 2        heteroatoms selected from O, N and S, the heterocyclic rings        being optionally further substituted by one or more C₁₋₄ groups        (e.g. methyl) and wherein the S, when present, may be present as        S, SO or SO₂;    -   five and six membered saturated heterocyclic rings containing 1        or 2 heteroatoms selected from O, N and S, the heterocyclic        rings being optionally further substituted by one or more C₁₋₄        groups (e.g. methyl) and wherein the S, when present, may be        present as S, SO or SO₂;    -   oxo; and    -   six membered aryl and heteroaryl rings containing up to two        nitrogen ring members and being optionally substituted by one or        substituents selected from halogen, methyl and methoxy.

In one preferred sub-group of compounds, R³ is a carbocyclic orheterocyclic group R^(3a) selected from phenyl; C₃₋₆ cycloalkyl; fiveand six membered saturated non-aromatic heterocyclic rings containing upto two heteroatom ring members selected from N, O, S and SO₂; sixmembered heteroaryl rings containing one, two or three nitrogen ringmembers; and five membered heteroaryl rings having up to threeheteroatom ring members selected from N, O and S;

wherein each carbocyclic or heterocyclic group R^(3a) is optionallysubstituted by up to four, preferably up to three, and more preferablyup to two (e.g. one) substituents selected from amino; hydroxy; oxo;fluorine; chlorine; C₁₋₄ alkyl-(O)_(q)— wherein q is 0 or 1 and the C₁₋₄alkyl moiety is optionally substituted by fluorine, hydroxy or C₁₋₂alkoxy; mono-C₁₋₄ alkylamino; di-C₁₋₄ alkylamino; C₁₋₄ alkoxycarbonyl;carboxy; a group R^(e)—R¹⁶ where R^(e) is a bond or a C₁₋₃ alkylenechain and R¹⁶ is selected from C₁₋₄ alkylsulphonyl; C₁₋₄alkylaminosulphonyl; C₁₋₄ alkylsulphonylamino-; amino; mono-C₁₋₄alkylamino; di-C₁₋₄ alkylamino; C₁₋₇-hydrocarbyloxycarbonylamino; sixmembered aromatic groups containing up to three nitrogen ring members;C₃₋₆ cycloalkyl; five or six membered saturated non-aromaticheterocyclic groups containing one or two heteroatom ring membersselected from N, O, S and SO₂, the group R¹⁶ when a saturatednon-aromatic group being optionally substituted by one or more methylgroups, and the group R¹⁶ when aromatic being optionally substituted byone or more groups selected from fluorine, chlorine, hydroxy, C₁₋₂alkoxyand C₁₋₂ alkyl.

In a further embodiment, R³ is selected from:

-   -   monocyclic aryl groups optionally substituted by 1-4 (for        example 1-2, e.g. 1) substituents R¹⁰ or R^(10a);    -   C₃-C₇ cycloalkyl groups optionally substituted by 1-4 (for        example 1-2, e.g. 1) substituents R¹⁰ or R^(10a);    -   saturated five membered heterocyclic rings containing 1 ring        heteroatom selected from O, N and S and being optionally        substituted by an oxo group and/or by 1-4 (for example 1-2,        e.g. 1) substituents R¹⁰ or R^(10a);    -   saturated six membered heterocyclic rings containing 1 or 2 ring        heteroatoms selected from O, N and S and being optionally        substituted by an oxo group and/or by 1-4 (for example 1-2,        e.g. 1) substituents R¹⁰ or R^(10a);    -   five membered heteroaryl rings containing 1 or 2 ring        heteroatoms selected from O, N and S and being optionally        substituted by 1-4 (for example 1-2, e.g. 1) substituents R¹⁰ or        R^(10a);    -   six membered heteroaryl rings containing 1 or 2 nitrogen ring        members (preferably 1 nitrogen ring member) and being optionally        substituted by 1-4 (for example 1-2, e.g. 1) substituents R¹⁰ or        R^(10a);    -   mono-azabicycloalkyl and diazabicycloalkyl groups each having 7        to 9 ring members and being optionally substituted by 1-4 (for        example 1-2, e.g. 1) substituents R¹⁰ or R^(10a).

Specific examples of the group Y—R³ are set out in Table 2. In Table 2,the point of attachment of the group to the nitrogen atom of thepyrazole-3-carboxamide group is represented by the terminal single bondextending from the group. Thus, by way of illustration, group CA in thetable is the 4-fluorophenyl, group CB in the table is the4-methoxybenzyl group and group CC in the table is the4-(4-methylpiperazino)-phenylmethyl group.

TABLE 2 Examples of the Group Y—R³

CA

CB

CC

CD

CE

CF

CG H CH

CI

CJ

CK

CL

CM

CN

CO

CP

CQ

CR

CS

CT

CU

CV

CW

CX

CY

CZ

DA

DB

DC

DD

DE

DF

DG

DH

DI

DJ

DK

DL

DM

DN

DO

DP

DQ

DR

DS

DT

DU

DV

DW

DX

DY

DZ

EA

EB

EC

ED

EE

EF

EG

EH

EI

EJ

EK

EL

EM

EN

EO

EP

EQ

ER

ES

ET

EU

EV

EW

EX

EY

EZ

FA

FB

FC

FD

FE

FF

FG

FH

FI

FJ

FK

FL

FM

FN

One sub-set of groups selected from table 2 consists of groups CA to EU.

Another sub-set of groups selected from table 2 consists of groups CA toCV.

Preferred groups selected from Table 2 include groups CL, CM, ES, ET,FC, FG and FH.

Particularly preferred groups selected from Table 2 include groups CL,CM and ES, and most preferably CL and CM.

In another general embodiment, when R³ is an aza-cycloalkyl group, thegroup X in the compound of the formula (I) is preferably R¹-A-NR⁴wherein A is CO, NR^(g)(C═O) or O(C═O). Additionally, or alternatively,when R³ is an aza-cycloalkyl group, the nitrogen atom of theaza-cycloalkyl group is preferably not substituted with an alkylenechain linked to a 2,3-dihydro-benzo[1,4]dioxine or tetrahydronaphthalenegroup.

In another general embodiment, when Y is an alkylene chain of 1 carbonatom in length, R³ is other than an optionally substituted phenyl groupbearing a substituted or unsubstituted cyclohexyloxy or cyclohexylthiogroup.

In another general embodiment, R³ is other than a moiety containing afive membered heteroaryl ring linked directly by a single bond to amonocyclic or bicyclic aryl group or R³ is other than a moietycontaining a bis heteroaryl group comprising two five memberedheteroaryl rings linked together by a single bond.

In a further general embodiment, R¹ is other than a moiety containing afive membered heteroaryl ring linked directly by a single bond to amonocyclic or bicyclic aryl group or R¹ is other than a moietycontaining a bis heteroaryl group comprising two five memberedheteroaryl rings linked together by a single bond.

In another general embodiment, R¹-A-NR⁴ is other than an optionallysubstituted nicotinoyl-amino or benzoyl-amino group when Y—R³ is analkyl, cycloalkyl, optionally substituted phenyl or optionallysubstituted phenylalkyl group.

When A is a bond (and optionally when A is CO, NR^(g)(C═O) or O(C═O)),Y—R³ may be other than a cycloalkyl group substituted at the 1-positionwith a hydrocarbon chain simultaneously bearing an oxy substituent suchas hydroxy, an aryl substituent and a diazole or triazole substituent.

Preferably, R¹ or R³ each are other than a moiety containing asubstituted phenyl group having thio and/or oxy substituents such ashydroxy, alkoxy and alkylthio at both the 3- and 4-positions of thephenyl ring.

In a further general embodiment, when Y—R³ is unsubstituted orsubstituted benzyl or phenethyl or naphthylmethyl, X may be other thanC₁₋₅ alkylamino or C₁₋₇ acylamino.

The group Y—R³ preferably does not include a benzo-fused lactam grouphaving attached thereto an unsubstituted or substituted imidazole group.

The group Y—R³ preferably does not include the moiety —CH═C(CO₂R^(q))—S—where R^(q) is hydrogen or alkyl.

In another general embodiment, neither R¹ nor R³ contain a moiety inwhich a five membered nitrogen-containing heteroaryl group is linkeddirectly or via an alkylene, oxa-alkylene, thia-alkylene or aza-alkylenegroup to an unsubstituted pyridyl group or to a substituted aryl,heteroaryl or piperidine ring, each said ring having attached thereto asubstituent selected from cyano, and substituted or unsubstituted amino,aminoalkyl, amidine, guanidine, and carbamoyl groups.

In a further general embodiment, R¹ and R³ are each other than anunsaturated nitrogen-containing heterocyclic group or anitrogen-containing heteroaryl group, or a benzfuran or benzthiophenegroup wherein the said nitrogen-containing heterocyclic group,nitrogen-containing heteroaryl group, bicyclic benzfuran orbenzthiophene group are linked directly by a single bond to asubstituted pyridyl or phenyl group.

In another general embodiment, neither R¹ nor R³ contain a moiety inwhich a five membered nitrogen-containing heteroaryl group is linkeddirectly or via an alkylene, oxa-alkylene, thia-alkylene or aza-alkylenegroup to a substituted aryl, heteroaryl or piperidine group or to anunsubstituted pyridyl group.

In general, it is preferred that the compounds of the invention, wherethey contain a carboxylic acid group, contain no more than one suchgroup.

Particular and Preferred Sub-Groups of the formulae (I), (Ia) and (Ib)

One particular group of compounds of the invention is represented by theformula (II):

or salts or tautomers or N-oxides or solvates thereof;

wherein R¹, R², R³ and Y are each independently selected from R¹, R², R³and Y as defined herein.

Within formula (II), it is preferred that R² is hydrogen or C₁₋₄ alkyl(e.g. C₁₋₃ alkyl), and more preferably R² is hydrogen.

In one sub-group of compounds of the formula (II), R¹ is:

phenyl optionally substituted by one or more substituents (e.g. 1, 2 or3) selected from fluorine; chlorine; hydroxy; 5- and 6-memberedsaturated heterocyclic groups containing 1 or 2 heteroatoms selectedfrom O, N and S, the heterocyclic groups being optionally substituted byone or more C₁₋₄ alkyl groups; C₁₋₄ hydrocarbyloxy; and C₁₋₄hydrocarbyl; wherein the C₁₋₄ hydrocarbyl and C₁₋₄ hydrocarbyloxy groupsare optionally substituted by one or more substituents chosen fromhydroxy, fluorine, C₁₋₂ alkoxy, amino, mono and di-C₁₋₄ alkylamino,phenyl, halophenyl, saturated carbocyclic groups having 3 to 7 ringmembers (more preferably 4, 5 or 6 ring members, e.g. 5 or 6 ringmembers) or saturated heterocyclic groups of 5 or 6 ring members andcontaining up to 2 heteroatoms selected from O, S and N; or 2,3-dihydro-benzo[1,4]dioxine; or

(ii) a monocyclic heteroaryl group containing one or two heteroatomsselected from O, S and N; or a bicyclic heteroaryl group containing asingle heteroatom selected from O, S and N; the monocyclic and bicyclicheteroaryl groups each being optionally substituted by one or moresubstituents selected from fluorine; chlorine; C₁₋₃ hydrocarbyloxy; andC₁₋₃ hydrocarbyl optionally substituted by hydroxy, fluorine, methoxy ora five or six membered saturated carbocyclic or heterocyclic groupcontaining up to two heteroatoms selected from O, S and N; or

(iii) a substituted or unsubstituted cycloalkyl group having from 3 to 6ring members; or

(iv) a C₁₋₄ hydrocarbyl group optionally substituted by one or moresubstituents selected from fluorine; hydroxy; C₁₋₄ hydrocarbyloxy;amino; mono- or di-C₁₋₄ hydrocarbylamino; and carbocyclic orheterocyclic groups having from 3 to 12 ring members, and wherein one ofthe carbon atoms of the hydrocarbyl group may optionally be replaced byan atom or group selected from O, NH, SO and SO₂.

Within group (i), a sub-group of groups R¹ consists of phenyl optionallysubstituted by one or more substituents selected from fluorine;chlorine; hydroxy; C₁₋₃ hydrocarbyloxy; and C₁₋₃ hydrocarbyl wherein theC₁₋₃ hydrocarbyl group is optionally substituted by one or moresubstituents chosen from hydroxy, fluorine, C₁₋₂ alkoxy, amino, mono anddi-C₁₋₄ alkylamino, saturated carbocyclic groups having 3 to 7 ringmembers (more preferably 4, 5 or 6 ring members, e.g. 5 or 6 ringmembers) or saturated heterocyclic groups of 5 or 6 ring members andcontaining up to 2 heteroatoms selected from O, S and N.

In another sub-group of compounds of the formula (II), R¹ is selectedfrom (i) and (iii) above and additionally from a sub-set (aii) wheresub-set (aii) consists of 2-furanyl, 3-furanyl, imidazolyl, 2-pyridyl,indolyl, 2-thienyl and 3-thienyl, each optionally substituted by one ormore substituents selected from fluorine, chlorine, C₁₋₃ hydrocarbyloxy,and C₁₋₃ hydrocarbyl optionally substituted by hydroxy, fluorine ormethoxy.

Within the group of compounds defined by the formula (II), where R¹ is(i) an optionally substituted phenyl group, it may be, for example, anunsubstituted phenyl group or a 2-monosubstituted, 3-monosubstituted,2,3 disubstituted, 2,5 disubstituted or 2,6 disubstituted phenyl groupor 2, 3-dihydro-benzo[1,4]dioxine, where the substituents are selectedfrom halogen; hydroxyl; C₁₋₃ alkoxy; and C₁₋₃ alkyl groups wherein theC₁₋₃ alkyl group is optionally substituted by hydroxy, fluorine, C₁₋₂alkoxy, amino, mono and di-C₁₋₄ alkylamino, or saturated carbocyclicgroups having 3 to 6 ring members and/or saturated heterocyclic groupsof 5 or 6 ring members and containing 1 or 2 heteroatoms selected from Nand O.

In one embodiment, R¹ is selected from unsubstituted phenyl,2-fluorophenyl, 2-hydroxyphenyl, 2-methoxyphenyl, 2-methylphenyl,2-(2-(pyrrolidin-1-yl)ethoxy)-phenyl, 3-fluorophenyl, 3-methoxyphenyl,2,6-difluorophenyl, 2-fluoro-6-hydroxyphenyl, 2-fluoro-3-methoxyphenyl,2-fluoro-5-methoxyphenyl, 2-chloro-6-methoxyphenyl,2-fluoro-6-methoxyphenyl, 2,6-dichlorophenyl and2-chloro-6-fluorophenyl, and is optionally further selected from5-fluoro-2-methoxyphenyl.

In another embodiment, R¹ is selected from unsubstituted phenyl,2-fluorophenyl, 2-hydroxyphenyl, 2-methoxyphenyl, 2-methylphenyl,2-(2-(pyrrolidin-1-yl)ethoxy)-phenyl, 3-fluorophenyl, 3-methoxyphenyl,2,6-difluorophenyl, 2-fluoro-6-hydroxyphenyl, 2-fluoro-3-methoxyphenyland 2-fluoro-5-methoxyphenyl.

Particular groups R¹ are 2,6-difluorophenyl, 2-fluoro-6-methoxyphenyland 2,6-dichlorophenyl.

One particularly preferred group R¹ is 2,6-difluorophenyl.

Another particularly preferred group R¹ is 2,6-dichlorophenyl.

When R¹ is (ii) a monocyclic heteroaryl group containing one or twoheteroatoms selected from O, S and N or a bicyclic heteroaryl groupcontaining a single heteroatom, examples of monocyclic and bicyclicheteroaryl groups include furanyl (e.g. 2-furanyl and 3-furanyl),imidazolyl, pyridyl (e.g. 2-pyridyl), indolyl, thienyl (e.g. 2-thienyland 3-thienyl) groups. The optional substituents for such groups caninclude chlorine, fluorine, methyl, methoxy, hydroxymethyl,methoxymethyl, morpholinomethyl, piperazinomethyl,N-methylypiperazinomethyl and piperidinylmethyl groups. Particularexamples of groups (ii) include unsubstituted 2-furanyl,3-methyl-2-furanyl, unsubstituted 4-(1H)-imidazolyl, unsubstituted5-(1H)-imidazolyl, unsubstituted 3-furanyl, unsubstituted 3-thienyl,2-methyl-3-thienyl and unsubstituted 3-pyrrolyl, and further examplesinclude 4-methoxy-3-thienyl, 5-(1-pyrrolidinyl)methyl-2-furyl and5-(4-morpholino)methyl-2-furyl groups.

When R¹ is (iii) an optionally substituted cycloalkyl group, it can befor example a substituted or unsubstituted cyclopropyl, cyclobutyl,cyclopentyl or cyclohexyl group. When the cycloalkyl group issubstituted, preferred substituents include methyl, fluorine andhydroxyl. Particular examples of cycloalkyl groups include1-methylcyclopropyl, 1-hydroxycyclopropyl, and unsubstituted cyclohexyl,cyclopentyl and cyclobutyl.

In the context of formula (II) and the group R¹, examples of optionallysubstituted hydrocarbyl groups are optionally substituted methyl, ethyland propyl groups wherein one of the carbon atoms of the hydrocarbylgroup is optionally replaced by O, NH, SO or SO₂. Particular examples ofsuch groups include methyl, ethyl, trifluoromethyl, methyl and ethylsubstituted with a carbocyclic or heterocyclic group having from 3 to 12ring members, sulphonylmethyl substituted with a carbocyclic orheterocyclic group having from 3 to 12 ring members, hydroxymethyl,hydroxyethyl, 3-hydroxy-2-propyl, propyl, isopropyl, butyl and tertiarybutyl. Examples of hydrocarbyl groups and carbocylic and heteroacyclicgroups are as set out above in the general definitions of such groups.Particular carbocyclic and heterocyclic groups include unsubstituted orsubstituted phenyl, indolyl, tetrazolyl, triazolyl, piperidinyl,morpholinyl, piperazinyl, N-methylpiperazinyl, imidazolyl wherein theoptional substituents may be selected from the group R¹⁰, and sub-groupsthereof, as defined herein.

In another sub-group of compounds of the formula (II), R¹ is a C₁₋₄hydrocarbyl group optionally substituted by one or more substituentsselected from fluorine, hydroxy, C₁₋₄ hydrocarbyloxy, amino, mono- ordi-C₁₋₄ hydrocarbylamino, and carbocyclic or heterocyclic groups havingfrom 3 to 12 ring members, and wherein 1 of the carbon atoms of thehydrocarbyl group may optionally be replaced by an atom or groupselected from O, NH, SO and SO₂.

In one embodiment, R¹ is a group R^(1a)—(V)_(n)— where:

n is 0 or 1;

V is selected from CH₂, CH₂CH₂ and SO₂CH₂; and

R^(1a) is a carbocyclic or heterocyclic group selected from phenyl;

five membered heteroaryl rings having up to 4 heteroatom ring membersselected from N, O and S;

six membered heteroaryl rings containing one or two nitrogen ringmembers;

five or six membered saturated non-aromatic heterocyclic ringscontaining one or two heteroatom ring members selected from N, O, S andSO₂;

C₃₋₆ cycloalkyl groups; indole; and quinoline;

wherein each of the carbocyclic and heterocyclic groups R^(1a) can beoptionally substituted by one or more substituents selected from five orsix membered saturated non-aromatic carbocyclic and heterocyclic groupscontaining up to two heteroatom ring members selected from N, O, S andSO₂; hydroxy; amino; oxo; mono-C₁₋₄ alkylamino; di-C₁₋₄ alkylamino;fluorine; chlorine; nitro; C₁₋₄ alkyl-(O)_(q)— wherein q is 0 or 1 andthe C₁₋₄ alkyl moiety is optionally substituted by fluorine, hydroxy,C₁₋₂alkoxy or a five or six membered saturated non-aromatic carbocyclicor heterocyclic group containing up to two heteroatom ring membersselected from N, O, S and SO₂; phenyl and C₁₋₂-alkylene dioxy.

Specific examples of groups R¹—CO— in formula (II) are set out in Table1 above.

One sub-group of preferred groups R¹—CO consists of the groups J, AB,AH, AJ, AL, AS, AX, AY, AZ, BA, BB, BD, BH, BL, BQ and BS.

Another sub-group of groups R¹—CO consists of the groups A to BF.

A further sub-group of groups R¹—CO consists of the groups A to BS.

Particularly preferred groups are the groups AJ, BQ and BS in Table 1,e.g. the sub-set consisting of AJ and BQ.

Another group of compounds of the invention is represented by theformula (III):

or salts or tautomers or N-oxides or solvates thereof;

wherein R¹, R², R³ and Y are as defined herein.

Examples of, and preferences, for the groups R¹, R², R³ and Y are as setout above for compounds of the formulae (0), (I⁰), (I), (Ia), (Ib) and(II) unless the context indicates otherwise.

Particular sub-groups of compounds of the formula (III) include:

compounds wherein R¹ is a heteroaryl group containing 1, 2 or 3heteroatom ring members selected from N, O and S;

(ii) compounds wherein R¹ is a C₁₋₆ hydrocarbyl group optionallysubstituted by one or more substituents selected from fluorine, hydroxy,C₁₋₄ hydrocarbyloxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, andcarbocyclic or heterocyclic groups having from 3 to 12 ring members, andwherein 1 of the carbon atoms of the hydrocarbyl group may optionally bereplaced by an atom or group selected from O, NH, SO and SO₂; and

(iii) compounds wherein R¹ is a non-aromatic carbocyclic or heterocyclicgroup having from 3 to 12 ring members.

Examples of compounds of the formula (III) wherein R¹ is (i) aheteroaryl group include 5- and 6-membered monocyclic heteroaryl groups,e.g. containing for 2 heteratom ring members selected from O, N and S.In one embodiment, the heteroaryl group is a monocyclic group containing1 or 2 nitrogen ring members. In another embodiment, the heteroarylgroups are selected from 6-membered rings containing 1 or 2 nitrogenring members, for example pyridine, pyrimidine, pyrazine and pridazinegroups, one particular sub-group consisting of pyrazinyl and pyridyl.

The heteroaryl groups can be unbsubstituted or substituted by one ormore groups R¹⁰ as defined herein.

Examples of compounds of the formula (III) wherein R¹ is (ii) anoptionally substituted C₁₋₆ hydrocarbyl group include those in which thehydrocarbyl group is unsubstituted hydrocarbyl, for exampleunsubstituted alkyl such as methyl, ethyl, propyl, isopropyl, butyl,isobutyl, t-butyl, 1-pentyl, 2-pentyl and 3-pentyl.

Examples of compounds wherein R¹ is a non-aromatic carbocyclic orheterocyclic group include those wherein the carbocyclic or heterocylicgroup is monocyclic and contains up to 2 heteroatoms selected fromoxygen and nitrogen. Particular examples of such groups are cyclohexyland piperidino.

Another sub-group of compounds of the formula (I) can be represented bythe formula (IV):

or salts or tautomers or N-oxides or solvates thereof;

wherein R¹ and R² are as defined herein;

an optional second bond may be present between carbon atoms numbered 1and 2;

one of U and T is selected from CH₂, CHR¹³, CR¹¹R¹³, NR¹⁴, N(O)R¹⁵, Oand S(O)_(t); and the other of U and T is selected from, NR¹⁴, O, CH₂,CHR¹¹, C(R¹¹)₂, and C═O; r is 0, 1, 2, 3 or 4; t is 0, 1 or 2;

R¹¹ is selected from hydrogen, halogen (particularly fluorine), C₁₋₃alkyl (e.g. methyl) and C₁₋₃ alkoxy (e.g. methoxy);

R¹³ is selected from hydrogen, NHR¹⁴, NOH, NOR¹⁴ and R^(a)—R^(b);

R¹⁴ is selected from hydrogen and R^(d)—R^(b);

R^(d) is selected from a bond, CO, C(X²)X¹, SO₂ and SO₂NR^(c);

R^(a), R^(b) and R^(c) are as hereinbefore defined; and

R¹⁵ is selected from C₁₋₄ saturated hydrocarbyl optionally substitutedby hydroxy, C₁₋₂ alkoxy, halogen or a monocyclic 5- or 6-memberedcarbocyclic or heterocyclic group, provided that U and T cannot be Osimultaneously.

Examples of, and preferences, for the groups R¹ and R² are as set outabove for compounds of the formulae (I), (Ia), (Ib) and (II) unless thecontext indicates otherwise.

Within formula (IV), r can be 0, 1, 2, 3 or 4. In one embodiment, r is0. In another embodiment, r is 2, and in a further embodiment r is 4.

Within formula (IV), one sub-set of preferred compounds is the set ofcompounds where there is only a single bond between the carbon atomsnumbered 1 and 2.

However, in another sub-set of compounds, there is a double bond betweenthe carbon atoms numbered 1 and 2.

Another sub-set of compounds is characterised by gem disubstitution atthe 2-carbon (when there is a single bond between carbon atoms numbers 1and 2) and/or the 6-carbon. Preferred gem disubstituents includedifluoro and dimethyl.

A further sub-set of compounds is characterised by the presence of analkoxy group, for example a methoxy group at the carbon atom numbered 3,i.e. at a position α with respect to the group T.

Within formula (IV) are compounds wherein, for example, R³ is selectedfrom any of the following ring systems:

Preferred ring systems include G1 and G3.

A preferred sub-group of compounds within formula (IV) can berepresented by the formula (IVa):

or salts or tautomers or N-oxides or solvates thereof;

wherein R¹ and R² are as hereinbefore defined;

one of U and T is selected from CH₂, CHR¹³, CR¹¹R¹³, NR¹⁴, N(O)R¹⁵, Oand S(O)_(t); and the other of U and T is selected from CH₂, CHR¹¹,C(R¹¹)₂, and C═O; r is 0, 1 or 2; t is 0, 1 or 2;

R¹¹ is selected from hydrogen and C₁₋₃ alkyl;

R¹³ is selected from hydrogen and R^(a)—R^(b);

R¹⁴ is selected from hydrogen and R^(d)—R^(b);

R^(d) is selected from a bond, CO, C(X²)X¹, SO₂ and SO₂NR^(c);

R^(a), R^(b) and R^(c) are as hereinbefore defined; and

R¹⁵ is selected from C₁₋₄ saturated hydrocarbyl optionally substitutedby hydroxy, C₁₋₂ alkoxy, halogen or a monocyclic 5- or 6-memberedcarbocyclic or heterocyclic group.

Examples of, and preferences, for the groups R¹ and R² are as set outabove for compounds of the formulae (0), (I⁰), (I), (Ia), (Ib) and (II)unless the context indicates otherwise.

In formula (IVa), T is preferably selected from CH₂, CHR¹³, CR¹¹R¹³,NR¹⁴, N(O)R¹⁵, O and S(O)_(t); and U is preferably selected from CH₂,CHR¹¹, C(R¹¹)₂, and C═O.

In the definitions for substituents R¹¹ and R¹⁴, R^(b) is preferablyselected from hydrogen; monocyclic carbocyclic and heterocyclic groupshaving from 3 to 7 ring members; and C₁₋₄ hydrocarbyl (more preferablyacyclic saturated C₁₋₄ groups) optionally substituted by one or moresubstituents selected from hydroxy, oxo, halogen, amino, mono- ordi-C₁₋₄ hydrocarbylamino, and monocyclic carbocyclic and heterocyclicgroups having from 3 to 7 ring members (more preferably 3 to 6 ringmembers) and wherein one or more carbon atoms of the C₁₋₄ hydrocarbylgroup may optionally be replaced by O, S, SO, SO₂, NR^(c), X¹C(X²),C(X²)X¹;

R^(c) is selected from hydrogen and C₁₋₄ hydrocarbyl; and

-   -   X¹ is O, S or NR^(c) and X² is ═O, ═S or ═NR^(c).

R¹¹ is preferably selected from hydrogen and methyl and most preferablyis hydrogen.

R¹³ is preferably selected from hydrogen; hydroxy; halogen; cyano;amino; mono-C₁₋₄ saturated hydrocarbylamino; di-C₁₋₄ saturatedhydrocarbylamino; monocyclic 5- or 6-membered carbocyclic andheterocyclic groups; C₁₋₄ saturated hydrocarbyl optionally substitutedby hydroxy, C₁₋₂ alkoxy, halogen or a monocyclic 5- or 6-memberedcarbocyclic or heterocyclic group.

Particular examples of R¹³ are hydrogen, hydroxy, amino, C₁₋₂ alkylamino(e.g. methylamino) C₁₋₄ alkyl (e.g. methyl, ethyl, propyl and butyl),C₁₋₂ alkoxy (e.g. methoxy), C₁₋₂ alkylsulphonamido (e.g.methanesulphonamido), hydroxy-C₁₋₂ alkyl (e.g. hydroxymethyl),C₁₋₂-alkoxy-C₁₋₂ alkyl (e.g. methoxymethyl and methoxyethyl), carboxy,C₁₋₄ alkoxycarbonyl (e.g. ethoxycarbonyl) and amino-C₁₋₂-alkyl (e.g.aminomethyl).

Particular examples of R¹⁴ are hydrogen; C₁₋₄ alkyl optionallysubstituted by fluoro or a five or six membered saturated heterocyclicgroup (e.g. a group selected from (i) methyl, ethyl, n-propyl, i-propyl,butyl, 2,2,2-trifluoroethyl and tetrahydrofuranylmethyl; and/or (ii)2-fluoroethyl and 2,2-difluoroethyl); cyclopropylmethyl; substituted orunsubstituted pyridyl-C₁₋₂ alkyl (e.g. 2-pyridylmethyl); substituted orunsubstituted phenyl-C₁₋₂ alkyl (e.g. benzyl); C₁₋₄ alkoxycarbonyl(e.g.ethoxycarbonyl and t-butyloxycarbonyl); substituted andunsubstituted phenyl-C₁₋₂ alkoxycarbonyl (e.g. benzyloxycarbonyl);substituted and unsubstituted 5- and 6-membered heteroaryl groups suchas pyridyl (e.g. 2-pyridyl and 6-chloro-2-pyridyl) and pyrimidinyl (e.g.2-pyrimidinyl); C₁₋₂-alkoxy-C₁₋₂ alkyl (e.g. methoxymethyl andmethoxyethyl); C₁₋₄ alkylsulphonyl (e.g. methanesulphonyl).

Preferred compounds include those in which (i) U is CHR¹³ (morepreferably CH₂) and T is NR¹⁴, and (ii) T is CHR¹³ (more preferably CH₂)and U is NR¹⁴.

One particular preferred sub-group of compounds of the formula (IV) canbe represented by the formula (Va):

or salts or tautomers or N-oxides or solvates thereof;

wherein R^(14a) is selected from hydrogen, C₁₋₄ alkyl optionallysubstituted by fluoro (e.g. methyl, ethyl, n-propyl, i-propyl, butyl and2,2,2-trifluoroethyl), cyclopropylmethyl, phenyl-C₁₋₂ alkyl (e.g.benzyl), C₁₋₄ alkoxycarbonyl (e.g.ethoxycarbonyl andt-butyloxycarbonyl), phenyl-C₁₋₂ alkoxycarbonyl (e.g.benzyloxycarbonyl), C₁₋₂-alkoxy-C₁₋₂ alkyl (e.g. methoxymethyl andmethoxyethyl), and C₁₋₄ alkylsulphonyl (e.g. methanesulphonyl), whereinthe phenyl moieties when present are optionally substituted by one tothree substituents selected from fluorine, chlorine, C₁₋₄ alkoxyoptionally substituted by fluoro or C₁₋₂-alkoxy, and C₁₋₄ alkyloptionally substituted by fluoro or C₁₋₂-alkoxy;

w is 0, 1, 2 or 3;

R² is hydrogen or methyl, most preferably hydrogen;

R¹¹ and r are as hereinbefore defined; and

R¹⁹ is selected from fluorine; chlorine; C₁₋₄ alkoxy optionallysubstituted by fluoro or C₁₋₂-alkoxy; and C₁₋₄ alkyl optionallysubstituted by fluoro or C₁₋₂-alkoxy.

Another particular preferred sub-group of compounds of the formula (IV)can be represented by the formula (Vb):

or salts or tautomers or N-oxides or solvates thereof;

wherein R^(14a) is selected from hydrogen, C₁₋₄ alkyl optionallysubstituted by fluoro (e.g. methyl, ethyl, n-propyl, i-propyl, butyl and2,2,2-trifluoroethyl), cyclopropylmethyl, phenyl-C₁₋₂ alkyl (e.g.benzyl), C₁₋₄ alkoxycarbonyl (e.g.ethoxycarbonyl andt-butyloxycarbonyl), phenyl-C₁₋₂ alkoxycarbonyl (e.g.benzyloxycarbonyl), C₁₋₂-alkoxy-C₁₋₂ alkyl (e.g. methoxymethyl andmethoxyethyl), and C₁₋₄ alkylsulphonyl (e.g.methanesulphonyl), whereinthe phenyl moieties when present are optionally substituted by one tothree substituents selected from fluorine, chlorine, C₁₋₄ alkoxyoptionally substituted by fluoro or C₁₋₂-alkoxy, and C₁₋₄ alkyloptionally substituted by fluoro or C₁₋₂-alkoxy;

w is 0, 1, 2 or 3;

R² is hydrogen or methyl, most preferably hydrogen;

R¹¹ and r are as hereinbefore defined; and

R¹⁹ is selected from fluorine; chlorine; C₁₋₄ alkoxy optionallysubstituted by fluoro or C₁₋₂-alkoxy; and C₁₋₄ alkyl optionallysubstituted by fluoro or C₁₋₂-alkoxy.

In formulae (Va) and (Vb), when w is 1, 2 or 3, it is preferred that thephenyl ring is 2-monosubstituted, 3-monosubstituted, 2,6-disubstituted,2,3-disubstituted, 2,4-disubstituted 2,5-disubstituted,2,3,6-trisubstituted or 2,4,6-trisubstituted. Most preferably the phenylring is disubstituted at positions 2- and 6- with substituents selectedfrom fluorine, chlorine and methoxy.

R¹¹ is preferably hydrogen (or r is 0).

R^(14a) is most preferably hydrogen or methyl.

One preferred sub-group of compounds of the formula (Va) can berepresented by the formula (VIa):

or salts or tautomers or N-oxides or solvates thereof;

wherein R²⁰ is selected from hydrogen and methyl;

R²¹ is selected from fluorine and chlorine; and

R²² is selected from fluorine, chlorine and methoxy; or

one of R²¹ and R²² is hydrogen and the other is selected from chlorine,methoxy, ethoxy, difluoromethoxy, trifluoromethoxy and benzyloxy.

Another preferred sub-group of compounds of the formula (Va) can berepresented by the formula (VIb):

or salts or tautomers or N-oxides or solvates thereof;

wherein R²⁰ is selected from hydrogen and methyl;

R^(21a) is selected from fluorine and chlorine; and

R^(22a) is selected from fluorine, chlorine and methoxy.

Particular compounds within formula (VIb) include:

4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide;

4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(1-methyl-piperidin-4-yl)-amide;

4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide; and

4-(2-fluoro-6-methoxy-benzoylamino)-1 H-pyrazole-3-carboxylic acidpiperidin-4-ylamide;

or salts or tautomers or N-oxides or solvates thereof.

A further group of compounds of the invention is represented by theformula (VII):

or salts or tautomers or N-oxides or solvates thereof;

wherein R², R³ and Y are as hereinbefore defined and G is a 5- or6-membered carbocyclic or heterocyclic ring.

The group G can be an unsubstituted carbocyclic or heterocyclic ring orit can be a substituted carbocyclic or heterocyclic ring bearing one ormore substituents selected from the groups R¹⁰ and R^(10a) ashereinbefore defined

The carbocyclic or heterocyclic ring may be aromatic or non-aromatic andexamples of such heterocyclic rings are set out above. In the context ofthe group G, preferred heterocyclic rings are those containing anitrogen ring atom through which the group G is connected to thepyrazole ring. Particular heterocyclic rings are saturated heterocyclicrings containing up to 3 nitrogen atoms (more usually up to 2, forexample 1) and optionally an oxygen atom. Particular examples of suchrings are six membered rings such as piperidine, piperazine, N-methylpiperazine and morpholine.

When the group G is a carbocyclic group, it can be, for example a6-membered aryl ring. For example, the group G can be an unsubstitutedphenyl group or it can be a substituted phenyl group bearing one or moresubstituents selected from the groups R¹⁰ and R^(10a) as hereinbeforedefined. The substituents, when present, are more typically smallsubstituents such as hydroxyl, halogen (e.g. fluorine and chlorine), andC₁₋₄ hydrocarbyl (methyl, ethyl and cyclopropyl) optionally substitutedby fluorine (e.g. trifluoromethyl) or hydroxy (e.g. hydroxymethyl).

In one general embodiment, when X is a non-aromatic heterocyclic group,then R³ may be other than a six membered monocyclic aryl or heteroarylgroup linked directly to a 5,6-fused bicyclic heteroaryl group.

A further group of compounds of the invention is represented by theformula (VIII):

or salts or tautomers or N-oxides or solvates thereof;

wherein R¹, R², R³ and Y are as defined herein.

Preferred groups R¹, R², Y and R³ are as set out above in the sectionheaded “General Preferences and Definitions” and in relation tocompounds of the formulae (I) and (II) and sub-groups thereof as definedherein.

For the avoidance of doubt, it is to be understood that each general andspecific preference, embodiment and example of the groups R¹ may becombined with each general and specific preference, embodiment andexample of the groups R² and/or R³ and/or R⁴ and/or R¹⁰ and/or Y and/orR^(g) and/or sub-groups thereof as defined herein and that all suchcombinations are embraced by this application.

The various functional groups and substituents making up the compoundsof the formula (I) are typically chosen such that the molecular weightof the compound of the formula (I) does not exceed 1000. More usually,the molecular weight of the compound will be less than 750, for exampleless than 700, or less than 650, or less than 600, or less than 550.More preferably, the molecular weight is less than 525 and, for example,is 500 or less.

Particular compounds of the formulae (0), (I⁰), (I), (Ia), (Ib), (II),(III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof are as illustrated in the examples below.

One particularly preferred compound is4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide and salts therof, particularly acid addition saltssuch as the methanesulphonic acid, acetic acid and hydrochloric acidsalts.

Salts, Solvates, Tautomers, Isomers, N-Oxides, Esters, Prodrugs andIsotopes

A reference to a particular cytotoxic compound or signalling inhibitoror compound of the formulae (0), (I⁰), (I), (Ia), (Ib), (II), (III),(IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groupsthereof also includes ionic, salt, solvate, isomers, tautomers,N-oxides, ester, prodrugs, isotopes and protected forms thereof, forexample, as discussed below. Preferably, the salts or tautomers orisomers or N-oxides or solvates thereof. More preferably, the salts ortautomers or N-oxides or solvates thereof.

Many compounds of the formula (I) can exist in the form of salts, forexample acid addition salts or, in certain cases salts of organic andinorganic bases such as carboxylate, sulphonate and phosphate salts. Allsuch salts are within the scope of this invention, and references tocompounds of the formula (I) include the salt forms of the compounds. Asin the preceding sections of this application, all references to formula(I) should be taken to refer also to formulae (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof unless the context indicates otherwise.

Salt forms may be selected and prepared according to methods describedin Pharmaceutical Salts: Properties, Selection, and Use, P. HeinrichStahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8,Hardcover, 388 pages, August 2002.

Acid addition salts may be formed with a wide variety of acids, bothinorganic and organic. Examples of acid addition salts include saltsformed with an acid selected from the group consisting of acetic,2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic),L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+)camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric,caproic, caprylic, carbonic, cinnamic, citric, cyclamic,dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic,2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic,glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic(e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic,hydrochloric, hydriodic, isethionic, (+)-L-lactic, (±)-DL-lactic,lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic,methanesulphonic, naphthalene-2-sulphonic, naphthalene-1,5-disulphonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic,4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic,(+)-L-tartaric, thiocyanic, p-toluenesulphonic, undecylenic and valericacids, as well as acylated amino acids and cation exchange resins.

One particular group of salts includes salts formed with an acidselected from the group consisting of acetic, adipic, alginic, ascorbic(e.g. L-ascorbic), aspartic (e.g. L-aspartic), benzenesulphonic,benzoic, camphoric (e.g. (+) camphoric), capric, caprylic, carbonic,citric, cyclamic, dodecanoate, dodecylsulphuric, ethane-1,2-disulphonic,ethanesulphonic, fumaric, galactaric, gentisic, glucoheptonic,D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic),α-oxoglutaric, glycolic, hippuric, hydrochloric, isethionic, isobutyric,lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic,laurylsulphonic, maleic, malic, (−)-L-malic, malonic, methanesulphonic,mucic, naphthalenesulphonic (e.g. naphthalene-2-sulphonic),naphthalene-1,5-disulphonic, nicotinic, oleic, orotic, oxalic, palmitic,pamoic, phosphoric, propionic, sebacic, stearic, succinic, sulphuric,tartaric (e.g. (+)-L-tartaric), thiocyanic, toluenesulphonic (e.g.p-toluenesulphonic), valeric and xinafoic acids.

Another particular group of salts consists of salts formed fromhydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulphonic,toluenesulphonic, methanesulphonic, ethanesulphonic,naphthalenesulphonic, valeric, acetic, propanoic, butanoic, malonic,glucuronic and lactobionic acids.

One preferred group of salts consists of salts formed frommethanesulphonic, hydrochloric, acetic, adipic, L-aspartic and DL-lacticacids.

Particular salts are salts formed with hydrochloric, methanesulphonicand acetic acids.

One preferred salt is the salt formed with methanesulphonic acid.

Another preferred salt is the salt formed with acetic acid.

A further preferred salt is the salt formed with hydrochloric acid.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the compounds of the invention contain an amine function, thesemay form quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of formula (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof as defined herein.

The salt forms of the compounds of the invention are typicallypharmaceutically acceptable salts, and examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19. However, saltsthat are not pharmaceutically acceptable may also be prepared asintermediate forms which may then be converted into pharmaceuticallyacceptable salts. Such non-pharmaceutically acceptable salts forms,which may be useful, for example, in the purification or separation ofthe compounds of the invention, also form part of the invention.

Particular salts for use in the preparation of liquid (e.g. aqueous)compositions of the compounds of formulae (I) and sub-groups andexamples thereof as described herein are salts having a solubility in agiven liquid carrier (e.g. water) of greater than 25 mg/ml of the liquidcarrier (e.g. water), more typically greater than 50 mg/ml andpreferably greater than 100 mg/ml.

In one embodiment of the invention, the compound of the formula (I) asdefined herein is provided in the form of a pharmaceutical compositioncomprising an aqueous solution containing the said compound in the formof a salt in a concentration of greater than 25 mg/ml, typically greaterthan 50 mg/ml and preferably greater than 100 mg/ml.

Compounds of the formula (I) containing an amine function may also formN-oxides. A reference herein to a compound of the formula (I) thatcontains an amine function also includes the N-oxide.

Where a compound contains several amine functions, one or more than onenitrogen atom may be oxidised to form an N-oxide. Particular examples ofN-oxides are the N-oxides of a tertiary amine or a nitrogen atom of anitrogen-containing heterocycle.

N-Oxides can be formed by treatment of the corresponding amine with anoxidizing agent such as hydrogen peroxide or a per-acid (e.g. aperoxycarboxylic acid), see for example Advanced Organic Chemistry, byJerry March, 4^(th) Edition, Wiley Interscience, pages. Moreparticularly, N-oxides can be made by the procedure of L. W. Deady (Syn.Comm. 1977, 7, 509-514) in which the amine compound is reacted withm-chloroperoxybenzoic acid (MCPBA), for example, in an inert solventsuch as dichloromethane.

Compounds of the formula (I) may exist in a number of differentgeometric isomeric, and tautomeric forms and references to compounds ofthe formula (I) include all such forms. For the avoidance of doubt,where a compound can exist in one of several geometric isomeric ortautomeric forms and only one is specifically described or shown, allothers are nevertheless embraced by formula (I).

For example, in compounds of the formula (I) the pyrazole group may takeeither of the following two tautomeric forms A and B. For simplicity,the general formula (I) illustrates form A but the formula is to betaken as embracing both tautomeric forms.

Other examples of tautomeric forms include, for example, keto-, enol-,and enolate-forms, as in, for example, the following tautomeric pairs:keto/enol (illustrated below), imine/enamine, amide/imino alcohol,amidine/amidine, nitroso/oxime, thioketone/enethiol, andnitro/aci-nitro.

Where compounds of the formula (I) contain one or more chiral centres,and can exist in the form of two or more optical isomers, references tocompounds of the formula (I) include all optical isomeric forms thereof(e.g. enantiomers, epimers and diastereoisomers), either as individualoptical isomers, or mixtures (e.g. racemic mixtures) or two or moreoptical isomers, unless the context requires otherwise.

The optical isomers may be characterised and identified by their opticalactivity (i.e. as + and − isomers, or d and I isomers) or they may becharacterised in terms of their absolute stereochemistry using the “Rand S” nomenclature developed by Cahn, Ingold and Prelog, see AdvancedOrganic Chemistry by Jerry March, 4^(th) Edition, John Wiley & Sons, NewYork, 1992, pages 109-114, and see also Cahn, Ingold & Prelog, Angew.Chem. Int. Ed. Engl., 1966, 5, 385-415.

Optical isomers can be separated by a number of techniques includingchiral chromatography (chromatography on a chiral support) and suchtechniques are well known to the person skilled in the art.

As an alternative to chiral chromatography, optical isomers can beseparated by forming diastereoisomeric salts with chiral acids such as(+)-tartaric acid, (−)-pyroglutamic acid, (−)-di-toluoyl-L-tartaricacid, (+)-mandelic acid, (−)-malic acid, and (−)-camphorsulphonic,separating the diastereoisomers by preferential crystallisation, andthen dissociating the salts to give the individual enantiomer of thefree base.

Where compounds of the formula (I) exist as two or more optical isomericforms, one enantiomer in a pair of enantiomers may exhibit advantagesover the other enantiomer, for example, in terms of biological activity.Thus, in certain circumstances, it may be desirable to use as atherapeutic agent only one of a pair of enantiomers, or only one of aplurality of diastereoisomers. Accordingly, the invention providescompositions containing a compound of the formula (I) having one or morechiral centres, wherein at least 55% (e.g. at least 60%, 65%, 70%, 75%,80%, 85%, 90% or 95%) of the compound of the formula (I) is present as asingle optical isomer (e.g. enantiomer or diastereoisomer). In onegeneral embodiment, 99% or more (e.g. substantially all) of the totalamount of the compound of the formula (I) may be present as a singleoptical isomer (e.g. enantiomer or diastereoisomer).

The compounds of the invention include compounds with one or moreisotopic substitutions, and a reference to a particular element includeswithin its scope all isotopes of the element. For example, a referenceto hydrogen includes within its scope ¹H, ²H (D), and ³H (T). Similarly,references to carbon and oxygen include within their scope respectively¹²C, ¹³C and ¹⁴C and ¹⁶O and ¹⁸O.

The isotopes may be radioactive or non-radioactive. In one embodiment ofthe invention, the compounds contain no radioactive isotopes. Suchcompounds are preferred for therapeutic use. In another embodiment,however, the compound may contain one or more radioisotopes. Compoundscontaining such radioisotopes may be useful in a diagnostic context.

Esters such as carboxylic acid esters and acyloxy esters of thecompounds of formula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV),(IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereofas defined herein bearing a carboxylic acid group or a hydroxyl groupare also embraced by Formula (I). Examples of esters are compoundscontaining the group -C(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Particular examples of esters aregroups include, but are not limited to, —C(═O)OCH₃, —C(═O)OCH₂CH₃,—C(═O)OC(CH₃)₃, and —C(═O)OPh. Examples of acyloxy (reverse ester)groups are represented by —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Particular examples ofacyloxy groups include, but are not limited to, —OC(═O)CH₃ (acetoxy),—OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Also encompassed by formula (I) are any polymorphic forms of thecompounds, solvates (e.g. hydrates), complexes (e.g. inclusion complexesor clathrates with compounds such as cyclodextrins, or complexes withmetals) of the compounds, and pro-drugs of the compounds. By “prodrugs”is meant for example any compound that is converted in vivo into abiologically active compound of the formula (I).

For example, some prodrugs are esters of the active compound (e.g., aphysiologically acceptable metabolically labile ester). Duringmetabolism, the ester group (—C(═O)OR) is cleaved to yield the activedrug. Such esters may be formed by esterification, for example, of anyof the carboxylic acid groups (—C(═O)OH) in the parent compound, with,where appropriate, prior protection of any other reactive groups presentin the parent compound, followed by deprotection if required.

Examples of such metabolically labile esters include those of theformula —C(═O)OR wherein R is:

C₁₋₇alkyl

(e.g., -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu);

C₁₋₇aminoalkyl

(e.g., aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4-morpholino)ethyl);and

acyloxy-C₁₋₇alkyl

(e.g., acyloxymethyl;

acyloxyethyl;

pivaloyloxymethyl;

acetoxymethyl;

1-acetoxyethyl;

1-(1-methoxy-1-methyl)ethyl-carbonxyloxyethyl;

1-(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl;

1-isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl;

1-cyclohexyl-carbonyloxyethyl;

cyclohexyloxy-carbonyloxymethyl;

1-cyclohexyloxy-carbonyloxyethyl;

(4-tetrahydropyranyloxy)carbonyloxymethyl;

1-(4-tetrahydropyranyloxy)carbonyloxyethyl;

(4-tetrahydropyranyl)carbonyloxymethyl; and

1-(4-tetrahydropyranyl)carbonyloxyethyl).

Also, some prodrugs are activated enzymatically to yield the activecompound, or a compound which, upon further chemical reaction, yieldsthe active compound (for example, as in ADEPT, GDEPT, LIDEPT, etc.). Forexample, the prodrug may be a sugar derivative or other glycosideconjugate, or may be an amino acid ester derivative.

Methanesulphonic acid and acetic acid Addition Salts of Compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide

The combinations of the invention may comprise any of the compounds,salts, solvates, tautomers and isotopes thereof and, where the contextadmits, N-oxides, other ionic forms and prodrugs, as described below.

References to the compound 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acid piperidin-4-ylamide and its acid additionsalts include within their scope all solvates, tautomers and isotopesthereof and, where the context admits, N-oxides, other ionic forms andprodrugs.

The acid addition salt may be selected from salts formed with an acidselected from the group consisting of acetic, adipic, alginic, ascorbic(e.g. L-ascorbic), aspartic (e.g. L-aspartic), benzenesulphonic,benzoic, camphoric (e.g. (+) camphoric), capric, caprylic, carbonic,citric, cyclamic, dodecanoate, dodecylsulphuric, ethane-1,2-disulphonic,ethanesulphonic, fumaric, galactaric, gentisic, glucoheptonic,D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic),α-oxoglutaric, glycolic, hippuric, isethionic, isobutyric, lactic (e.g.(+)-L-lactic and (±)-DL-lactic), lactobionic, laurylsulphonic, maleic,malic, (−)-L-malic, malonic, methanesulphonic, mucic,naphthalenesulphonic (e.g. naphthalene-2-sulphonic),naphthalene-1,5-disulphonic, nicotinic, oleic, orotic, oxalic, palmitic,pamoic, phosphoric, propionic, sebacic, stearic, succinic, sulphuric,tartaric (e.g. (+)-L-tartaric), thiocyanic, toluenesulphonic (e.g.p-toluenesulphonic), valeric and xinafoic acids.

One sub-group of acid addition salts includes salts formed with an acidselected from the group consisting of acetic, adipic, ascorbic (e.g.L-ascorbic), aspartic (e.g. L-aspartic), caproic, carbonic, citric,dodecanoic, fumaric, galactaric, glucoheptonic, gluconic (e.g.D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic),glycolic, hippuric, lactic (e.g. (+)-L-lactic and (±)-DL-lactic),maleic, palmitic, phosphoric, sebacic, stearic, succinic, sulphuric,tartaric (e.g. (+)-L-tartaric) and thiocyanic acids.

More particularly the salts are acid addition salts formed with an acidselected from methanesulphonic acid and acetic acid, and mixturesthereof.

In one embodiment, the salt is an acid addition salt formed withmethanesulphonic acid.

In another embodiment, the salt is an acid addition salt formed withacetic acid.

For convenience the salts formed from methanesulphonic acid and aceticacid may be referred to herein as the methanesulphonate or mesylatesalts and acetate salts respectively.

In the solid state, the salts can be crystalline or amorphous or amixture thereof.

In one embodiment, the salts are amorphous.

In an amorphous solid, the three dimensional structure that normallyexists in a crystalline form does not exist and the positions of themolecules relative to one another in the amorphous form are essentiallyrandom, see for example Hancock et al. J. Pharm. Sci. (1997), 86, 1).

In another embodiment, the salts are substantially crystalline; i.e.they are from 50% to 100% crystalline, and more particularly they may beat least 50% crystalline, or at least 60% crystalline, or at least 70%crystalline, or at least 80% crystalline, or at least 90% crystalline,or at least 95% crystalline, or at least 98% crystalline, or at least99% crystalline, or at least 99.5% crystalline, or at least 99.9%crystalline, for example 100% crystalline.

In a further embodiment, the salts are selected from the groupconsisting of salts that are from 50% to 100% crystalline, salts thatare at least 50% crystalline, salts that are at least 60% crystalline,salts that are at least 70% crystalline, salts that are at least 80%crystalline, salts that are at least 90% crystalline, salts that are atleast 95% crystalline, salts that are at least 98% crystalline, saltsthat are at least 99% crystalline, salts that are at least 99.5%crystalline, and salts that are at least 99.9% crystalline, for example100% crystalline.

More preferably the salts may be those (or may be selected from thegroup consisting of those) that are 95% to 100% crystalline, for exampleat least 98% crystalline, or at least 99% crystalline, or at least 99.5%crystalline, or at least 99.6% crystalline or at least 99.7% crystallineor at least 99.8% crystalline or at least 99.9% crystalline, for example100% crystalline.

One example of a substantially crystalline salt is a crystalline saltformed with methanesulphonic acid.

Another example of a substantially crystalline salt is a crystallinesalt formed with acetic acid.

The salts, in the solid state, can be solvated (e.g. hydrated) ornon-solvated (e.g. anhydrous).

In one embodiment, the salts are non-solvated (e.g. anhydrous). Anexample of a non-solvated salt is the crystalline salt formed withmethanesulphonic acid as defined herein.

The term “anhydrous” as used herein does not exclude the possibility ofthe presence of some water on or in the salt (e.g a crystal of thesalt). For example, there may be some water present on the surface ofthe salt (e.g. salt crystal), or minor amounts within the body of thesalt (e.g. crystal). Typically, an anhydrous form contains fewer than0.4 molecules of water per molecule of compound, and more preferablycontains fewer than 0.1 molecules of water per molecule of compound, forexample 0 molecules of water.

In another embodiment, the salts are solvated. Where the salts arehydrated, they can contain, for example, up to three molecules of waterof crystallisation, more usually up to two molecules of water, e.g. onemolecule of water or two molecules of water. Non-stoichiometric hydratesmay also be formed in which the number of molecules of water present isless than one or is otherwise a non-integer. For example, where there isless than one molecule of water present, there may be for example 0.4,or 0.5, or 0.6, or 0.7, or 0.8, or 0.9 molecules of water present permolecule of compound.

Other solvates include alcoholates such as ethanolates andisopropanolates.

The salts can be synthesized from the parent compound4-(2,6-dichloro-benzoylamino)-1 H-pyrazole-3-carboxylic acidpiperidin-4-ylamide by conventional chemical methods such as methodsdescribed in Pharmaceutical Salts: Properties, Selection, and Use, P.Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN:3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such saltscan be prepared by reacting the parent compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide with the appropriate acid in water or in an organicsolvent, or in a mixture of the two; generally, nonaqueous media such asether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

One method of preparing an acid addition salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide comprises forming a solution of4-(2,6-dichloro-benzoylamino)-1 H-pyrazole-3-carboxylic acidpiperidin-4-ylamide free base in a solvent (typically an organicsolvent) or mixture of solvents, and treating the solution with an acidto form a precipitate of the acid addition salt.

The acid may be added as a solution in a solvent which is miscible withthe solvent in which the free base is dissolved. The solvent in whichthe free base is initially dissolved may be one in which the acidaddition salt thereof is insoluble. Alternatively, the solvent in whichthe free base is initially dissolved may be one in which the acidaddition salt is at least partially soluble, a different solvent inwhich the acid addition salt is less soluble subsequently being addedsuch that the salt precipitates out of solution.

In an alternative method of forming an acid addition salt,4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide is dissolved in a solvent comprising a volatile acidand optionally a co-solvent, thereby to form a solution of the acidaddition salt with the volatile acid, and the resulting solution is thenconcentrated or evaporated to isolate the salt. An example of an acidaddition salt that can be made in this way is the acetate salt.

In another aspect, the combination of the invention includes an acidaddition salt of 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid piperidin-4-ylamide as defined herein, obtained (or obtainable) bytreating a compound of the formula (X):

with an organic or inorganic acid as defined herein, other thanhydrochloric acid, in an organic solvent to remove thetert-butyloxycarbonyl group and form an acid addition salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide with the organic or inorganic acid, and optionallyisolating the acid addition salt thus formed.

The salt is typically precipitated from the organic solvent as it isformed and hence can be isolated by separation of the solid from thesolution, e.g. by filtration.

One salt form can be converted to the free base and optionally toanother salt form by methods well known to the skilled person. Forexample, the free base can be formed by passing the salt solutionthrough a column containing an amine stationary phase (e.g. a Strata-NH₂column). Alternatively, a solution of the salt in water can be treatedwith sodium bicarbonate to decompose the salt and precipitate out thefree base. The free base may then be combined with another acid by oneof the methods described above or elsewhere herein.

The methanesulphonate salt form is particularly advantageous because ofits good stability at elevated temperatures and in conditions of highrelative humidity, its non-hygroscopicity (as defined herein), absenceof polymorph and hydrate formation, and stability in aqueous conditions.Moreover, it has excellent water solubility and has betterphysiochemical properties (such as a high melting point) relative toother salts.

The term ‘stable’ or ‘stability’ as used herein includes chemicalstability and solid state (physical) stability. The term ‘chemicalstability’ means that the compound can be stored in an isolated form, orin the form of a formulation in which it is provided in admixture withfor example, pharmaceutically acceptable carriers, diluents or adjuvantsas described herein, under normal storage conditions, with little or nochemical degradation or decomposition. ‘Solid-state stability’ means thecompound can be stored in an isolated solid form, or the form of a solidformulation in which it is provided in admixture with, for example,pharmaceutically acceptable carriers, diluents or adjuvants as describedherein, under normal storage conditions, with little or no solid-statetransformation (e.g. hydration, dehydration, solvatisation,desolvatisation, crystallisation, recrystallisation or solid-state phasetransition).

The terms “non-hygroscopic” and “non-hygroscopicity” and related termsas used herein refer to substances that absorb less than 5% by weight(relative to their own weight) of water when exposed to conditions ofhigh relative humidity, for example 90% relative humidity, and/or do notundergo changes in crystalline form in conditions of high humidityand/or do not absorb water into the body of the crystal (internal water)in conditions of high relative humidity.

Preferred salts for use in the combinations of the invention are acidaddition salts (such as the mesylate and acetate and mixtures thereof asdefined herein) having a solubility in a given liquid carrier (e.g.water) of greater than 15 mg/ml of the liquid carrier (e.g. water), moretypically greater than 20 mg/ml, preferably greater than 25 mg/ml, andmore preferably greater than 30 mg/ml.

In another aspect, there is provided a combination comprising an aqueoussolution containing an acid addition salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (such as the mesylate and acetate and mixturesthereof as defined herein, and preferably the mesylate) in aconcentration of greater than 15 mg/ml, typically greater than 20 mg/ml,preferably greater than 25 mg/ml, and more preferably greater than 30mg/ml.

In a preferred embodiment, the combination comprises an aqueous solutioncontaining an acid addition salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide selected from an acetate or methanesulphonate saltor a mixture thereof in a concentration of greater than 15 mg/ml,typically greater than 20 mg/ml, preferably greater than 25 mg/ml, andmore preferably greater than 30 mg/ml.

In another aspect, the combination of the invention includes an aqueoussolution of an acid addition salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (such as the mesylate and acetate and mixturesthereof as defined herein), wherein the aqueous solution has a pH of 2to 12, for example 2 to 9, and more particularly 4 to 7.

In the aqueous solutions defined above, the acid addition salt may beany of the salts described herein but, in one preferred embodiment, is amesylate or acetate salt as defined herein, and in particular themesylate salt.

The combinations of the invention may include an aqueous solution of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide in protonated form together with one or more counterions and optionally one or more further counter ions. In one embodimentone of the counter ions is selected from methanesulphonate and acetate.In another embodiment one of the counter ions is from the formulationbuffer as described herein such as acetate. In a further embodimentthere may be one or more further counter ions such as a chloride ion(e.g. from saline).

The combinations of the invention may include an aqueous solution of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide in protonated form together with one or more counterions selected from methanesulphonate and acetate and optionally one ormore further counter ions such as a chloride ion.

In the situation where there is more than one counter ion the aqueoussolution of 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide in protonated form will potentially contain amixture of counter ions for example a mixture of methanesulphonate andacetate counter ions and optionally one or more further counter ionssuch as a chloride ion.

The combinations of the invention may include an aqueous solution of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide in protonated form together with one or more counterions selected from methanesulphonate and acetate and optionally one ormore further counter ions such as a chloride ion, and a mixture thereof.

The aqueous solutions can be formed inter alia by dissolving a mesylatesalt in a solution of acetate ions (e.g an acetate buffer) or bydissolving an acetate salt in a solution of mesylate ions. The mesylateand acetate ions may be present in the solution in a mesylate:acetateratio of from 10:1 or less, for example 10:1 to 1:10, more preferablyless then 8:1, or less than 7:1, or less than 6:1, or less than 5:1 orless than 4:1 or less than 3:1 or less than 2:1 or less than 1:1, moreparticularly from 1:1 to 1:10. In one embodiment, the mesylate andacetate ions are present in the solution in a mesylate:acetate ratio offrom 1:1 to 1:10, for example 1:1 to 1:8, or 1:1 to 1:7 or 1:1 to 1:6 or1:1 to 1:5, e.g. approximately 1:4.8.

The aqueous solutions of the salts may be buffered or unbuffered but inone embodiment are buffered.

In the context of the acid addition salt formed with methanesulphonicacid, a preferred buffer is a buffer formed from acetic acid and sodiumacetate, for example at a solution pH of approximately 4.6. At this pHand in the acetate buffer, the methanesulphonic acid salt has asolubility of about 35 mg/ml.

The salts for use in the combinations of the invention are typicallypharmaceutically acceptable salts, and examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19. However, saltsthat are not pharmaceutically acceptable may also be prepared asintermediate forms which may then be converted into pharmaceuticallyacceptable salts. Such non-pharmaceutically acceptable salt formstherefore also form part of the invention.

Biological Activity

The cytotoxic compounds and signalling inhibitors of the combinations ofthe invention interfere with metabolic processes vital to the physiologyand proliferation of cancer cells as described above and have activityagainst various cancers.

The compounds of the formulae (0), (I⁰), (I), (Ia), (Ib), (II), (III),(IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groupsthereof are inhibitors or modulators (in particular inhibitors) of oneor more cyclin dependent kinases and/or glycogen synthase kinases, andin particular one or more cyclin dependent kinases selected from CDK1,CDK2, CDK3, CDK4, CDK5, CDK6 and CDK9, and more particularly selectedfrom CDK1, CDK2, CDK3, CDK4, CDK5 and CDK9.

Preferred compounds of the formulae (0), (I⁰), (I), (Ia), (Ib), (II),(III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof are compounds that inhibit one or more CDK kinasesselected from CDK1, CDK2, CDK4 and CDK9, for example CDK1 and/or CDK2.

The compounds of the formulae (0), (I⁰), (I), (Ia), (Ib), (II), (III),(IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groupsthereof may modulate or inhibit GSKs such as glycogen synthase kinase-3(GSK3).

As a consequence of their activity in modulating or inhibiting CDKkinases and/or glycogen synthase kinases, and the activity of thecytotoxic agents and signalling inhibitors described herein, thecombinations of the invention are expected to be useful in providing ameans of arresting, or recovering control of, the cell cycle inabnormally dividing cells. It is therefore anticipated that thecompounds will prove useful in treating or preventing proliferativedisorders such as cancers.

CDKs play a role in the regulation of the cell cycle, apoptosis,transcription, differentiation and CNS function. Therefore, CDKinhibitors could be useful in the treatment of diseases in which thereis a disorder of proliferation, apoptosis or differentiation such ascancer. In particular RB+ve tumours may be particularly sensitive to CDKinhibitors. RB-ve tumours may also be sensitive to CDK inhibitors.

Examples of cancers which may be inhibited include, but are not limitedto, a carcinoma, for example a carcinoma of the bladder, breast, colon(e.g. colorectal carcinomas such as colon adenocarcinoma and colonadenoma), kidney, epidermis, liver, lung, for example adenocarcinoma,small cell lung cancer and non-small cell lung carcinomas, oesophagus,gall bladder, ovary, pancreas e.g. exocrine pancreatic carcinoma,stomach, cervix, thyroid, prostate, or skin, for example squamous cellcarcinoma; a hematopoietic tumour of lymphoid lineage, for exampleleukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, orBurkett's lymphoma; a hematopoietic tumour of myeloid lineage, forexample acute and chronic myelogenous leukemias, myelodysplasticsyndrome, or promyelocytic leukemia; thyroid follicular cancer; a tumourof mesenchymal origin, for example fibrosarcoma or habdomyosarcoma, atumour of the central or peripheral nervous system, for exampleastrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma;teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoctanthoma;thyroid follicular cancer; Kaposi's sarcoma, B-cell lymphoma and chroniclymphocytic leukaemia.

The cancers may be cancers which are sensitive to inhibition of any oneor more cyclin dependent kinases selected from CDK1, CDK2, CDK3, CDK4,CDK5 and CDK6, for example, one or more CDK kinases selected from CDK1,CDK2, CDK4 and CDK5, e.g. CDK1 and/or CDK2.

Whether or not a particular cancer is one which is sensitive toinhibition by a cyclin dependent kinase may be determined by means of acell growth assay as set out in the examples below or by a method as setout in the section headed “Methods of Diagnosis”.

Thus, in the pharmaceutical compositions, uses or methods of thisinvention for treating a disease or condition comprising abnormal cellgrowth, the disease or condition comprising abnormal cell growth in oneembodiment is a cancer.

One group of cancers includes human breast cancers (e.g. primary breasttumours, node-negative breast cancer, invasive duct adenocarcinomas ofthe breast, non-endometrioid breast cancers); and mantle cell lymphomas.In addition, other cancers are colorectal and endometrial cancers.

Another sub-set of cancers includes breast cancer, ovarian cancer, coloncancer, prostate cancer, oesophageal cancer, squamous cancer andnon-small cell lung carcinomas.

A further sub-set of cancers includes non small cell lung cancer, coloncancer, breast cancer, non-hodgkin's lymphoma, multiple myeloma andchromic lymphocytic leukemia.

A yet further sub-set of cancers includes breast cancer, colorectalcancer, ovarian cancer and non-small cell lung carcinoma.

A yet further sub-set of cancers includes colorectal cancer, ovariancancer and non-small cell lung carcinoma.

Another sub-set of cancers includes hematopoietic tumours of lymphoidlineage, for example leukemia, chronic lymphocytic leukaemia, mantlecell lymphoma and B-cell lymphoma (such as diffuse large B celllymphoma).

One particular cancer is chronic lymphocytic leukaemia.

Another particular cancer is mantle cell lymphoma.

Another particular cancer is diffuse large B cell lymphoma.

The activity of the compounds of the invention as inhibitors ormodulators of cyclin dependent kinases and/or glycogen synthase kinases(e.g. GSK-3) can be measured using the assays set forth in the examplesbelow and the level of activity exhibited by a given compound can bedefined in terms of the 1050 value. Preferred compounds of the presentinvention are compounds having an 1050 value of less than 1 micromole,more preferably less than 0.1 micromole.

Methods for the Preparation of Compounds of Formula (I) of the Invention

Compounds of the formula (I) and the various sub-groups thereof can beprepared in accordance with synthetic methods well known to the skilledperson. Unless stated otherwise, R¹, R², R³, Y, X and A are ashereinbefore defined.

In this section, as in all the other sections of this application,references to formula (I) should be taken to refer also to formulae (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof unless the contextindicates otherwise.

Compounds of the formula (I) wherein R¹-A- forms an acyl group R¹—CO—can be prepared by reacting a carboxylic acid of the formula R¹—CO₂H oran activated derivative thereof with an appropriately substituted4-amino-pyrazole as shown in Scheme 1.

The starting material for the synthetic route shown in Scheme 1 is the4-nitro-pyrazole-3-carboxylic acid (X) which can either be obtainedcommercially or can be prepared by nitration of the corresponding4-unsubstituted pyrazole carboxy compound.

The 4-nitro-pyrazole carboxylic acid (X), or a reactive derivativethereof, is reacted with the amine H₂N—Y—R³ to give the 4-nitro-amide(XI). The coupling reaction between the carboxylic acid (X) and theamine is preferably carried out in the presence of a reagent of the typecommonly used in the formation of peptide linkages. Examples of suchreagents include 1,3-dicyclohexylcarbodiimide (DCC) (Sheehan et al, J.Amer. Chem Soc. 1955, 77, 1067),1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (referred to hereineither as EDC or EDAC but also known in the art as EDCI and WSCDI)(Sheehan et al, J. Org. Chem., 1961, 26, 2525), uronium-based couplingagents such as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and phosphonium-based coupling agents such as1-benzo-triazolyloxytris-(pyrrolidino)phosphonium hexafluorophosphate(PyBOP) (Castro et al, Tetrahedron Letters, 1990, 31, 205).Carbodiimide-based coupling agents are advantageously used incombination with 1-hydroxy-7-azabenzotriazole (HOAt) (L. A. Carpino, J.Amer. Chem. Soc., 1993, 115, 4397) or 1-hydroxybenzotriazole (HOBt)(Konig et al, Chem. Ber., 103, 708, 2024-2034). Preferred couplingreagents include EDC (EDAC) and DCC in combination with HOAt or HOBt.

The coupling reaction is typically carried out in a non-aqueous,non-protic solvent such as acetonitrile, dioxan, dimethylsulphoxide,dichloromethane, dimethylformamide or N-methylpyrrolidine, or in anaqueous solvent optionally together with one or more miscibleco-solvents. The reaction can be carried out at room temperature or,where the reactants are less reactive (for example in the case ofelectron-poor anilines bearing electron withdrawing groups such assulphonamide groups) at an appropriately elevated temperature. Thereaction may be carried out in the presence of a non-interfering base,for example a tertiary amine such as triethylamine orN,N-diisopropylethylamine.

As an alternative, a reactive derivative of the carboxylic acid, e.g. ananhydride or acid chloride, may be used. Reaction with a reactivederivative such an anhydride is typically accomplished by stirring theamine and anhydride at room temperature in the presence of a base suchas pyridine.

Amines of the formula H₂N—Y—R³ can be obtained from commercial sourcesor can be prepared by any of a large number of standard syntheticmethods well known those skilled in the art, see for example seeAdvanced Organic Chemistry by Jerry March, 4^(th) Edition, John Wiley &Sons, 1992, and Organic Syntheses, Volumes 1-8, John Wiley, edited byJeremiah P. Freeman (ISBN: 0-471-31192-8), 1995, and see also themethods described in the experimental section below.

The nitro-pyrazole amide (XI) is reduced to give the corresponding4-amino-compound of the formula (XII). The reduction may be carried outby standard methods such as catalytic hydrogenation, for example in thepresence of palladium on carbon in a polar solvent such as ethanol ordimethylformamide at room temperature. As an alternative, reduction maybe effected using a reducing agent such as tin (II) chloride in ethanol,typically with heating, for example to the reflux temperature of thesolvent.

The 4-amino-pyrazole compound (XII) is then reacted with a carboxylicacid of the formula R¹—CO₂H, or a reactive derivative thereof, using themethods and conditions described above for the formation of the amide(XI), to give a compound of the formula (I).

Carboxylic acids of the formula R¹—CO₂H can be obtained commercially orcan be synthesised according to methods well known to the skilledperson, see for example Advanced Organic Chemistry and OrganicSyntheses, the details for which are given above.

Compounds of the formula (I) in which X is a group R¹-A-NR⁴, where A isa bond, can be prepared from the 4-amino compounds of the formula (XII)by a number of methods. Reductive amination with an appropriatelysubstituted aldehyde or ketone can be carried out in the presence ofvariety of reducing agents (see Advanced Organic Chemistry by JerryMarch, 4^(th) Edition, John Wiley & Sons, 1992, pp 898-900. For example,reductive amination can be carried out in the presence of sodiumtriacetoxyborohydride in the presence of an aprotic solvent such asdichloromethane at or near ambient temperatures.

Compounds in which X is a group R¹-A-NR⁴ where A is a bond can also beprepared by the reaction of the 4-amino pyrazole compound (XII) with acompound of the formula R¹-L in a nucleophilic displacement reactionwhere L is a leaving group such as a halogen.

In an alternative synthetic route, compounds of the formula (I) can beprepared by reaction of a compound of the formula (XIII) with a compoundof the formula R³—Y—NH₂. The reaction can be carried out using the amidecoupling conditions described above.

Compounds of the formula (I) where A is NH(C═O) can be prepared usingstandard methods for the synthesis of ureas. For example, such compoundscan be prepared by reacting an aminopyrazole compound of the formula(XII) with a suitably substituted phenylisocyanate in a polar solventsuch as DMF. The reaction is conveniently carried out at roomtemperature.

Compounds of the formula (I) where A is O(C═O) can be made usingstandard methods for the synthesis of carbamates, for example byreaction of an amino pyrazole compound of the formula (XII) with achloroformate derivative of the formula R¹—O—C(O)—Cl under conditionswell known to the skilled person.

Compounds of the formula (I), wherein A is SO₂, can be prepared fromamino-compounds of the formula (XII) by standard methods for theformation of sulphonamides. For example, compounds of the formula XII)can be reacted with sulphonyl chlorides of the formula R¹SO₂Cl oranhydrides of the formula (R¹SO₂)₂O. The reaction is typically carriedout in an aprotic solvent such as acetonitrile or a chlorinatedhydrocarbon (for example dichloromethane) in the presence of anon-interfering base such as a tertiary amine (e.g. triethylamine) orpyridine, or diisopropylethyl amine (Hunigs base). Alternatively, wherethe base is a liquid, as is the case with pyridine, the base itself maybe used as the solvent for the reaction.

Compounds wherein X is a 5- or 6-membered ring containing a carbon atomring member linked to the pyrazole group can be prepared by the sequenceof reactions set out in Scheme 2.

As shown in Scheme 2, an aldehyde (XIV) (in which X is a C-linked arylor heteroaryl group such as phenyl) is condensed with malononitrile togive the alkyne (XVI). The reaction is typically carried out in a polarsolvent such as ethanol in the presence of a base such as piperidine,usually with heating. The alkyne (XVI) is then reacted withtrimethylsilyldiazomethane in the presence an alkyl lithium such asbutyl lithium to give the 5-trimethylsilyl pyrazole-3-nitrile (XVII).The reaction is carried out in a dry aprotic solvent such as THF under aprotective atmosphere (e.g. nitrogen) at a reduced temperature (e.g. −78° C.).

The nitrile (XVII) is hydrolysed with an alkali metal hydroxide such aspotassium hydroxide to give the acid (XIX) and/or the amide (XVII).Where a mixture of acid and amide are formed, they may be separatedaccording to standard methods such as chromatography. The acid (XIX) canthen be coupled with an amine of the formula R³—Y—NH₂ under typicalamide coupling conditions of the type described above to give thecompound of the formula (I).

Alternatively, compounds of the formula (I) in which X is a C-linkedaryl or heteroaryl group such as phenyl can be prepared from compoundsof the formula (XX):

where “Hal” is a halogen such as chlorine, bromine or iodine, by meansof a Suzuki coupling reaction with the appropriate aryl or heteroarylboronate. The reaction can be carried out under typical Suzuki Couplingconditions in the presence of a palladium catalyst such asbis(tri-t-butylphosphine)palladium and a base (e.g. a carbonate such aspotassium carbonate). The reaction may be carried out in an aqueoussolvent system, for example aqueous ethanol, and the reaction mixture istypically subjected to heating, for example to a temperature in excessof 100° C.

Compounds of the formula (XX) can be prepared from amino-pyrazolecompounds of the formula (XII) by means of the Sandmeyer reaction (seeAdvanced Organic Chemistry, 4^(th) edition, by Jerry March, John Wiley &Sons, 1992, page 723) in which the amino group is converted to adiazonium group by reaction with nitrous acid, and the diazoniumcompound is then reacted with a copper (I) halide such as Cu(I)Cl orCu(I)I.

Once formed, one compound of the formula (I) may be transformed intoanother compound of the formula (I) using standard chemistry procedureswell known in the art. For examples of functional groupinterconversions, see for example, Fiesers' Reagents for OrganicSynthesis, Volumes 1-17, John Wiley, edited by Mary Fieser (ISBN:0-471-58283-2), and Organic Syntheses, Volumes 1-8, John Wiley, editedby Jeremiah P. Freeman (ISBN: 0-471-31192-8), 1995.

The starting materials for the synthetic routes shown in the Schemesabove, e.g. the pyrazoles of formula (X), can either be obtainedcommercially or can be prepared by methods known to those skilled in theart. They can be obtained using known methods e.g. from ketones, such asin a process described in EP308020 (Merck), or the methods discussed bySchmidt in Helv. Chim. Acta., 1956, 39, 986-991 and Helv. Chim. Acta.,1958, 41, 306-309. Alternatively they can be obtained by conversion of acommercially available pyrazole, for example those containing halogen,nitro, ester, or amide functionalities, to pyrazoles containing thedesired functionality by standard methods known to a person skilled inthe art. For example, in 3-carboxy-4-nitropyrazole, the nitro group canbe reduced to an amine by standard methods.4-Nitro-pyrazole-3-carboxylic acid (XII) can either be obtainedcommercially or can be prepared by nitration of the corresponding4-unsubstituted pyrazole carboxy compound, and pyrazoles containing ahalogen, may be utilized in coupling reactions with tin or palladiumchemistry.

Protecting Groups

In many of the reactions described above, it may be necessary to protectone or more groups to prevent reaction from taking place at anundesirable location on the molecule. Examples of protecting groups, andmethods of protecting and deprotecting functional groups, can be foundin Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rdEdition; John Wiley and Sons, 1999).

A hydroxy group may be protected, for example, as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a tetrahydropyranyl(THP) ether; a benzyl, benzhydryl (diphenylmethyl), or trityl(triphenylmethyl)ether; a trimethylsilyl or t-butyldimethylsilyl ether;or an acetyl ester (—OC(═O)CH₃, —OAc).

An aldehyde or ketone group may be protected, for example, as an acetal(R—CH(OR)₂) or ketal (R₂C(OR)₂), respectively, in which the carbonylgroup (>C═O) is converted to a diether (>C(OR)₂), by reaction with, forexample, a primary alcohol. The aldehyde or ketone group is readilyregenerated by hydrolysis using a large excess of water in the presenceof acid.

An amine group may be protected, for example, as an amide (—NRCO—R) or aurethane (—NRCO—OR), for example, as: a methyl amide (—NHCO—CH₃); abenzyloxy amide (—NHCO—OCH₂C₆H₅, —NH-Cbz or NH—Z); as a t-butoxy amide(—NHCO—OC(CH₃)₃, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), or as a2(-phenylsulphonyl)ethyloxy amide (—NH-Psec).

For example, in Scheme 1 above, when the moiety R³ in the amine H2N—Y—R³contains a second amino group, such as a cyclic amino group (e.g. apiperidine or pyrrolidine group), the second amino group can beprotected by means of a protecting group as hereinbefore defined, onepreferred group being the tert-butyloxycarbonyl (Boc) group. Where nosubsequent modification of the second amino group is required, theprotecting group can be carried through the reaction sequence to give anN-protected form of a compound of the formula (I) which can then bede-protected by standard methods (e.g. treatment with acid in the caseof the Boc group) to give the compound of formula (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein.

Other protecting groups for amines, such as cyclic amines andheterocyclic N—H groups, include toluenesulphonyl (tosyl) andmethanesulphonyl (mesyl) groups, benzyl groups such as apara-methoxybenzyl (PMB) group and tetrahydropyranyl (THP) groups.

A carboxylic acid group may be protected as an ester for example, as: anC₁₋₇ alkyl ester (e.g., a methyl ester; a t-butyl ester); a C₁₋₇haloalkyl ester (e.g., a C₁₋₇ trihaloalkyl ester); atriC₁₋₇alkylsilyl-C₁₋₇alkyl ester; or a C₅₋₂₀ aryl-C₁₋₇ alkyl ester(e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, forexample, as a methyl amide. A thiol group may be protected, for example,as a thioether (—SR), for example, as: a benzyl thioether; anacetamidomethyl ether (—S—CH₂NHC(═O)CH₃).

Isolation and Purification of the Compounds of the Invention

The compounds of the invention can be isolated and purified according tostandard techniques well known to the person skilled in the art. Onetechnique of particular usefulness in purifying the compounds ispreparative liquid chromatography using mass spectrometry as a means ofdetecting the purified compounds emerging from the chromatographycolumn.

Preparative LC-MS is a standard and effective method used for thepurification of small organic molecules such as the compounds describedherein. The methods for the liquid chromatography (LC) and massspectrometry (MS) can be varied to provide better separation of thecrude materials and improved detection of the samples by MS.Optimisation of the preparative gradient LC method will involve varyingcolumns, volatile eluents and modifiers, and gradients. Methods are wellknown in the art for optimising preparative LC-MS methods and then usingthem to purify compounds. Such methods are described in Rosentreter U,Huber U.; Optimal fraction collecting in preparative LC/MS; J CombChem.; 2004; 6(2), 159-64 and Leister W, Strauss K, Wisnoski D, Zhao Z,Lindsley C., Development of a custom high-throughput preparative liquidchromatography/mass spectrometer platform for the preparativepurification and analytical analysis of compound libraries; J CombChem.; 2003; 5(3); 322-9.

An example of such a system for purifying compounds via preparativeLC-MS is described below in the Examples section of this application(under the heading “Mass Directed Purification LC-MS System”). However,it will be appreciated that alternative systems and methods to thosedescribed could be used. In particular, normal phase preparative LCbased methods might be used in place of the reverse phase methodsdescribed here. Most preparative LC-MS systems utilise reverse phase LCand volatile acidic modifiers, since the approach is very effective forthe purification of small molecules and because the eluents arecompatible with positive ion electrospray mass spectrometry. Employingother chromatographic solutions e.g. normal phase LC, alternativelybuffered mobile phase, basic modifiers etc as outlined in the analyticalmethods described below could alternatively be used to purify thecompounds.

Cytotoxic Compounds and Signalling Inhibitors for use According to theInvention

Any of a wide variety of cytotoxic compounds and signalling inhibitorsmay be used in the combinations of the invention. Cytotoxicity may beassayed or determined using any of a wide variety of techniqueswell-known to those skilled in the art. The cytotoxic compounds andsignalling inhibitors of the combinations of the invention have activityagainst various cancers.

Preferably, the cytotoxic compounds for use in the combinations of theinvention as described herein are selected from the following classes:

-   -   1. camptothecin compounds;    -   2. antimetabolites;    -   3. vinca alkaloids;    -   4. taxanes;    -   5. platinum compounds;    -   6. DNA binders and Topo II inhibitors (including anthracycline        derivatives);    -   7. a combination of two or more of the foregoing classes.

Suitable signalling inhibitors are discussed in section 7, below.

A reference to a particular cytotoxic compound or signalling inhibitorherein (for example, a reference to a camptothecin compound,antimetabolite, vinca alkaloid, taxane, platinum compound, DNA binder,Topo II inhibitor (including anthracycline derivatives)) is intended toinclude ionic, salt, solvate, isomers, tautomers, N-oxides, ester,prodrugs, isotopes and protected forms thereof (preferably the salts ortautomers or isomers or N-oxides or solvates thereof, and morepreferably, the salts or tautomers or N-oxides or solvates thereof).

1. Camptothecin Compounds

In one embodiment of the invention, the cytotoxic compound is acamptothecin compound.

Definition: The term “camptothecin compound” as used herein refers tocamptothecin per se or analogues of camptothecin as described herein,including the ionic, salt, solvate, isomers, tautomers, N-oxides, ester,prodrugs, isotopes and protected forms thereof (preferably the salts ortautomers or isomers or N-oxides or solvates thereof, and morepreferably, the salts or tautomers or N-oxides or solvates thereof), asdescribed above.

Technical background: Camptothecin compounds are compounds related to orderived from the parent compound camptothecin which is a water-insolublealkaloid derived from the Chinese tree Camptothecin acuminata and theIndian tree Nothapodytes foetida. Camptothecin has a potent inhibitoryactivity against DNA biosynthesis and has shown high activity againsttumour cell growth in various experimental systems. Its clinical use inanti-cancer therapy is, however, limited significantly by its hightoxicity, and various analogues have been developed in attempts toreduce the toxicity of camptothecin while retaining the potency of itsanti-tumour effect. Examples of such analogues include irinotecan andtopotecan.

These compounds have been found to be specific inhibitors of DNAtopoisomerase I. Topoisomerases are enzymes that are capable of alteringDNA topology in eukaryotic cells. They are critical for importantcellular functions and cell proliferation. There are two classes oftopoisomerases in eukaryotic cells, namely type I and type II.Topoisomerase I is a monomeric enzyme having a molecular weight ofapproximately 100,000. The enzyme binds to DNA and introduces atransient single-strand break, unwinds the double helix (or allows it tounwind) and subsequently reseals the break before dissociating from theDNA strand.

Irinotecan, namely7-ethyl-10-(4-(1-piperidino)-1-piperidino)carbonyloxy-(20S)-camptothecin,and its hydrochloride, also known as CPT 11, have been found to haveimproved potency and reduced toxicity, and superior water-solubility.Irinotecan has been found to have clinical efficacy in the treatment ofvarious cancers especially colorectal cancer. Another importantcamptothecin compound is topotecan, namely(S)-9-dimethylaminomethyl-10-hydroxy-camptothecin which, in clinicaltrials, has shown efficacy against several solid tumours, particularlyovarian cancer and non-small cell lung carcinoma.

Exemplary formulations: A parenteral pharmaceutical formulation foradministration by injection and containing a camptothecin compound canbe prepared by dissolving 100 mg of a water soluble salt of thecamptothecin compound (for example a compound as described in EP 0321122and in particular the examples therein) in 10 ml of sterile 0.9% salineand then sterilising the solution and filling the solution into asuitable container.

Biological activity: The camptothecin compounds of the combinations ofthe invention are specific inhibitors of DNA topoisomerase I aredescribed above and have activity against various cancers.

Prior art references: WO 01/64194 (Janssen) discloses combinations offarnesyl transferase inhibitors and camptothecin compounds. EP 137145(Rhone Poulenc Rorer) discloses camptothecin compounds includingirinotecan. EP 321122 (SmithKline Beecham) discloses camptothecincompounds including topotecan.

Problems: Although camptothecin compounds have widely used aschemotherapeutic agents in humans, they are not therapeuticallyeffective in all patients or against all types of tumours. There istherefore a need to increase the inhibitory efficacy of camptothecincompounds against tumour growth and also to provide a means for the useof lower dosages of camptothecin compounds to reduce the potential foradverse toxic side effects to the patient.

Preferences: Preferred camptothecin compounds for use in accordance withthe invention include irinotecan and topotecan referred to above.Irinotecan is commercially available for example from Rhone-PoulencRorer under the trade name “Campto” and may be prepared for example asdescribed in European patent specification No. 137145 or by processesanalogous thereto. Topotecan is commercially available for example fromSmithKline Beecham under the trade name “Hycamtin” and may be preparedfor example as described in European patent number 321122 or byprocesses analogous thereto. Other camptothecin compounds may beprepared in conventional manner for example by processes analogous tothose described above for irinotecan and topotecan.

Specific embodiments: In one embodiment, the camptothecin compound isirinotecan. In another embodiment, the camptothecin compound is acamptothecin compound other than irinotecan, for example a camptothecincompound such as topotecan.

Posology: The camptothecin compound is advantageously administered in adosage of 0.1 to 400 mg per square metre (mg/m²) of body surface area,for example 1 to 300 mg/m², particularly for irinotecan in a dosage ofabout 100 to 350 mg/m² and for topotecan in about 1 to 2 mg/m² percourse of treatment. These dosage may be administered for example once,twice or more per course of treatment, which may be repeated for exampleevery 7, 14, 21 or 28 days.

2. Antimetabolites

In another embodiment of the invention, the cytotoxic compound is anantimetabolite.

Definition: The terms “antimetabolic compound” and “antimetabolite” areused as synonyms and define antimetabolic compounds or analogues ofantimetabolic compounds as described herein, including the ionic, salt,solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopes andprotected forms thereof (preferably the salts or tautomers or isomers orN-oxides or solvates thereof, and more preferably, the salts ortautomers or N-oxides or solvates thereof), as described above. Thus,the antimetabolic compounds, otherwise known as antimetabolites,referred to herein consitute a large group of anticancer drugs thatinterfere with metabolic processes vital to the physiology andproliferation of cancer cells. Such compounds include nucleosidederivatives, either pyrimidine or purine nucleoside analogs, thatinhibit DNA synthesis, and inhibitors of thymidylate synthase and/ordihydrofolate reductase enzymes.

Technical background: Antimetabolites (or antimetabolic compounds),constitute a large group of anticancer drugs that interfere withmetabolic processes vital to the physiology and proliferation of cancercells. Such compounds include nucleoside derivatives, either pyrimidineor purine nucleoside analogues, that inhibit DNA synthesis, andinhibitors of thymidylate synthase and/or dihydrofolate reductaseenzymes. Anti-tumour nucleoside derivatives have been used for manyyears for the treatment of various cancers. Among the oldest and mostwidely used of these derivatives is 5-fluorouracil (5-FU) which has beenused to treat a number of cancers such as colorectal, breast, hepaticand head and neck tumours.

In order to enhance the cytotoxic effect of 5-FU, leucovorin has beenused with the drug to modulate levels of thymidylate synthase which arecritical to ensure that malignant cells are sensitive to the effect of5-FU.

However, various factors limit the use of 5-FU, for example tumourresistance, toxicities, including gastrointestinal and haematologicaleffects, and the need for intravenous administration. Various approacheshave been taken to overcome these disadvantages including proposals toovercome the poor bioavailability of 5-FU and also to increase thetherapeutic index of 5-FU, either by reducing systemic toxicity or byincreasing the amount of active drug reaching the tumour.

One such compound which provides improved therapeutic advantage over5-FU is capecitabine, which has the chemical name[1-(5-deoxy-β-D-ribofuranosyl)-5-fluoro-1,2-dihydro-2-oxo-4-pyrimidinyl]-carbamicacid pentyl ester. Capecitabine is a pro-drug of 5-FU which is wellabsorbed after oral dosing and delivers pharmacologically-activeconcentrations of 5-FU to tumours, with little systemic exposure to theactive drug. As well as offering potentially superior activity to 5-FU,it can also be used for oral therapy with prolonged administration.Another anti-tumour nucleoside derivative is gemcitabine which has thechemical name 2′-deoxy-2′,2′-difluoro-cytidine, and which has been usedin the treatment of various cancers including non-small cell lung cancerand pancreatic cancer. Further anti-tumour nucleosides includecytarabine and fludarabine. Cytarabine, also known as ara-C, which hasthe chemical name 1-β-D-arabinofuranosylcytosine, has been found usefulin the treatment of acute myelocytic leukemia, chronic myelocyticleukemia (blast phase), acute lymphocytic leukemia and erythroleukemia.

Fludarabine is a DNA synthesis inhibitor, which has the chemical name9-β-D-arabinofuranosyl-2-fluoro-adenine, and is used for the treatmentof refractory B-cell chronic lymphocytic leukaemia. Otherantimetabolites used in anticancer chemotherapy include the enzymeinhibitors raltitrexed, pemetrexed, and methotrexate.

Raltitrexed is a folate-based thymidylate synthase inhibitor, which hasthe chemical nameN-[5-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)-methyl-N-methylamino]-2-thenoyl]-L-glutamic acid, and is used in the treatmentof advanced colorectal cancer.

Pemetrexed is a thymidylate synthase and transferase inhibitor, whichhas the chemical nameN-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamicacid, disodium salt, and is used for the treatment of mesothelioma andlocally advanced or metastatic non-small-cell lung cancer (SCLC) inpreviously treated patients.

Methotrexate is an antimetabolite which interrupts cell division byinhibiting DNA replication through dihydrofolate reductase inhibition,resulting in cell death, and has the chemical name isN-[4-[[(2,4-diamino-6-pteridinyl)methyl]-ethylamino]benzoyl]-L-glutamicacid, and is used for the treatment of acute lymphocytic leukemia, andalso in the treatment of breast cancer, epidermoid cancers of the headand neck, and lung cancer, particularly squamous cell and small celltypes, and advanced stage non-Hodgkin's lymphomas.

Biological activity: The antimetabolic compounds of the combinations ofthe invention interfere with metabolic processes vital to the physiologyand proliferation of cancer cells as described above and have activityagainst various cancers.

Problems: These anticancer agents have a number of side-effectsespecially myelosuppression and in some cases nausea and diarrhoea.There is therefore a need to provide a means for the use of lowerdosages to reduce the potential of adverse toxic side effects to thepatient.

Preferences: Preferred antimetabolic compounds for use in accordancewith the invention include anti-tumour nucleosides such as5-fluorouracil, gemcitabine, capecitabine, cytarabine and fludarabineand enzyme inhibitors such as ralitrexed, pemetrexed and methotrexatereferred to herein. Thus, preferred antimetabolic compounds for use inaccordance with the invention are anti-tumour nucleoside derivativesincluding 5-fluorouracil, gemcitabine, capecitabine, cytarabine andfludarabine referred to herein. Other preferred antimetabolic compoundsfor use in accordance with the invention are enzyme inhibitors includingralitrexed, pemetrexed and methotrexate.

5-Fluorouracil is widely available commercially, or may be prepared forexample as described in U.S. Pat. No. 2,802,005. Gemcitabine iscommercially available for example from Eli Lilly and Company under thetrade name Gemzar, or may be prepared for example as described inEuropean patent specification No. 122707, or by processes analogousthereto. Capecitabine is commercially available for example fromHoffman-La Roche Inc under the trade name Xeloda, or may be prepared forexample as described in European patent specification No. 698611, or byprocesses analogous thereto. Cytarabine is commercially available forexample from Pharmacia and Upjohn Co under the trade name Cytosar, ormay be prepared for example as described in U.S. Pat. No. 3,116,282, orby processes analogous thereto. Fludarabine is commercially availablefor example from Schering AG under the trade name Fludara, or may beprepared for example as described in U.S. Pat. No. 4,357,324, or byprocesses analogous thereto. Ralitrexed is commercially available forexample from AstraZeneca plc under the trade name Tomudex, or may beprepared for example as described in European patent specification No.239632, or by processes analogous thereto. Pemetrexed is commerciallyavailable for example from Eli Lilly and Company under the trade nameAlimta, or may be prepared for example as described in European patentspecification No. 432677, or by processes analogous thereto.Methotrexate is commercially available for example from LederleLaboraories under the trade name Methotrexate-Lederle, or may beprepared for example as described in U.S. Pat. No. 2,512,572, or byprocesses analogous thereto. Other antimetabolites for use in thecombinations of the invention include 6-mercapto purine, 6-thioguanine,cladribine, 2′-deoxycoformycin and hydroxyurea.

Specific embodiments: In one embodiment, the antimetabolic compound isgemcitabine. In another embodiment, the antimetabolic compound is aantimetabolic compound other than 5-fluorouracil or fludarabine, forexample an antimetabolic compound such as gemcitabine, capecitabine,cytarabine, ralitrexed, pemetrexed or methotrexate.

Posology: The antimetabolite compound will be administered in a dosagethat will depend on the factors noted above. Examples of dosages forparticular preferred antimetabolites are given below by way of example.With regard to anti-tumour nucleosides, these are advantageouslyadministered in a daily dosage of 10 to 2500 mg per square meter (mg/m²)of body surface area, for example 700 to 1500 mg/m², particularly for5-FU in a dosage of 200 to 500 mg/m², for gemcitabine in a dosage of 800to 1200 mg/m², for capecitabine in a dosage of 1000 to 1200 mg/m², forcytarabine in a dosage of 100-200mg/m² and for fludarabine in a dosageof 10 to 50 mg/m².

For the following enzyme inhibitors, examples are given of possibledoses. Thus, raltitrexed can be administered in a dosage of about 3mg/m², pemetrexed in a dosage of 500 mg/m² and methotrexate in a dosageof 30-40 mg/m².

The dosages noted above may generally be administered for example once,twice or more per course of treatment, which may be repeated for exampleevery 7, 14, 21 or 28 days.

3. Vinca Alkaloids

In another embodiment of the invention, the cytotoxic compound is avinca alkaloid.

Definition: The term “vinca alkaloid” as used herein refers to vincaalkaloid compounds or analogues of vinca alkaloid compounds as describedherein, including the ionic, salt, solvate, isomers, tautomers,N-oxides, ester, prodrugs, isotopes and protected forms thereof(preferably the salts or tautomers or isomers or N-oxides or solvatesthereof, and more preferably, the salts or tautomers or N-oxides orsolvates thereof), as described above.

Technical background: The vinca alkaloids for use in the combinations ofthe invention are anti-tumour vinca alkaloids related to or derived fromextracts of the periwinkle plant (Vinca rosea). Among these compounds,vinblastine and vincristine are important clinical agents for thetreatment of leukaemias, lymphomas and testicular cancer, andvinorelbine has activity against lung cancer and breast cancer.

Biological activity: The vinca alkaloid compounds of the combinations ofthe invention are tubulin targeting agents and have activity againstvarious cancers, particularly a sub-set of cancers including leukaemias,lymphomas, testicular cancer, lung cancer and breast cancer.

Problems: Vinca alkaloids suffer from toxicological effects. Forexample, vinblastine causes leukopenia which reaches a nadir in 7 to 10days following drug administration, after which recovery ensues within 7days, while vincristine demonstrates some neurological toxicity forexample numbness and trembling of the extremities, loss of deep tendonreflexes and weakness of distal limb musculature. Vinorelbine has sometoxicity in the form of granulocytopenia but with only modestthrombocytopenia and less neurotoxicity than other vinca alkaloids.There is therefore a need to increase the inhibitory efficacy ofanti-tumour vinca alkaloids against tumour growth and also to provide ameans for the use of lower dosages of anti-tumour vinca alkaloids toreduce the potential of adverse toxic side effects to the patient.

Preferences: Preferred anti-tumour vinca alkaloids for use in accordancewith the invention include vindesine, vinvesir, vinblastine, vincristineand vinorelbine. Particularly preferred anti-tumour vinca alkaloids foruse in accordance with the invention include vinblastine, vincristineand vinorelbine refererred to above. Vinblastine is commerciallyavailable for example as the sulphate salt for injection from Eli Lillyand Co under the trade name Velban, and may be prepared for example asdescribed in German patent specification No. 2124023 or by processesanalogous thereto. Vincristine is commercially available for example asthe sulphate salt for injection from Eli Lilly and Co under the tradename Oncovin and may be prepared for example as described in the aboveGerman patent specification No. 2124023 or by processes analogousthereto. Vincristine is also available as a liposomal formulation underthe name Onco-TCS™. Vinorelbine is commercially available for example asthe tartrate salt for injection from Glaxo Wellcome under the trade nameNavelbine and may be prepared for example as described in U.S. Pat. No.4,307,100, or by processes analogous thereto. Other anti-tumour vincaalkaloids may be prepared in conventional manner for example byprocesses analogous to those described above for vinoblastine,vincristine and vinorelbine.

Another preferred vinca alkaloid is vindesine. Vindesine is a syntheticderivative of the dimeric catharanthus alkaloid vinblastine, isavailable from Lilly under the tradename Eldisine and from Shionogiunder the tradename Fildesin. Details of the synthesis of Vindesine aredescribed in Lilly patent DE2415980 (1974) and by C. J. Burnett et al.,J. Med. Chem. 21, 88 (1978).

Specific embodiments: In one embodiment, the vinca alkaloid compound isselected from vinoblastine, vincristine and vinorelbine. In anotherembodiment, the vinca alkaloid compound is vinoblastine.

Posology: The anti-tumour vinca alkaloid is advantageously administeredin a dosage of 2 to 30 mg pr square meter (mg/m²) of body surface area,particularly for vinblastine in a dosage of about 3 to 12 mg/m², forvincristine in a dosage of about 1 to 2 mg/m², and for vinorelbine indosage of about 10 to 30 mg/m² per course of treatment. These dosagesmay be administered for example once, twice or more per course oftreatment, which may be repeated for example every 1, 14, 21 or 28 days.

4. Taxanes

In another embodiment of the invention, the cytotoxic compound is ataxane.

Definition: The term “taxane compound” as used herein refers to taxanecompounds or analogues of taxane compounds as described herein,including the ionic, salt, solvate, isomers, tautomers, N-oxides, ester,prodrugs, isotopes and protected forms thereof (preferably the salts ortautomers or isomers or N-oxides or solvates thereof, and morepreferably, the salts or tautomers or N-oxides or solvates thereof), asdescribed above.

Technical background: The taxanes are a class of compounds having thetaxane ring system and related to or derived from extracts from certainspecies of yew (Taxus) trees. These compounds have been found to haveactivity against tumour cell growth and certain compounds in this classhave been used in the clinic for the treatment of various cancers. Thus,for example, paclitaxel is a diterpene isolated from the bark of the yewtree, Taxus brevifolia, and can be produced by partial synthesis from10-acetylbacctin, a precursor obtained from yew needles and twigs or bytotal synthesis, see Holton et al, J. Am. Chem. Soc. 116; 1597-1601(1994) and Nicholau et al, Nature 367:630 (1994). Paclitaxel has shownanti-neoplastic activity and more recently it has been established thatits antitumour activity is due to the promotion of microtubulepolymerisation, Kumar N. J., Biol. Chem. 256: 1035-1041 (1981); Rowinskyet al, J. Natl. Cancer Inst. 82: 1247-1259 (1990); and Schiff et al,Nature 277: 655-667 (1979). Paclitaxel has now demonstrated efficacy inseveral human tumours in clinical trials, McGuire et al, Ann. Int. Med.,111:273-279 (1989); Holmes et al, J. Natl. Cancer Inst. 83: 1797-1805(1991); Kohn et al J. Natl. Cancer Inst. 86: 18-24 (1994); and Kohn etal, American Society for Clinical Oncology, 12 (1993). Paclitaxel hasfor example been used for the treatment of ovarian cancer and alsobreast cancer.

Another taxane compound which has been used in the clinic is docetaxelwhich has been shown to have particular efficacy in the treatment ofadvanced breast cancer. Docetaxel has shown a better solubility inexcipient systems than paclitaxel, therefore increasing the ease withwhich it can be handled and used in pharmaceutical compositions.

Biological activity: The taxane compounds of the combinations of theinvention are tubulin targeting agents and have activity against variouscancers.

Problems: Clinical use of taxanes has demonstrated a narrow therapeuticindex with many patients unable to tolerate the side effects associatedwith its use. There is therefore a need to increase the inhibitoryefficacy of taxane compounds against tumour growth and also to provide ameans for the use of lower dosages of taxane compounds to reduce thepotential of adverse toxic side effects to the patient.

Preferences: Preferred taxane compounds for use in accordance with theinvention include paclitaxel or docetaxel referred to herein. Paclitaxelis available commercially for example under the trade name Taxol fromBristol Myers Squibb and docetaxel is available commercially under thetrade name Taxotere from Rhone-Poulenc Rorer. Both compounds and othertaxane compounds may be prepared in conventional manner for example asdescribed in EP 253738, EP 253739 and WO 92/09589 or by processesanalogous thereto.

Specific embodiments: In one embodiment, the taxane compound ispaclitaxel. In another embodiment, the taxane compound is docetaxel.

Posology: The taxane compound is advantageously administered in a dosageof 50 to 400 mg per square metere (mg/m²) of body surface area, forexample 75 to 250 mg/m², particularly for paclitaxel in a dosage ofabout 175 to 250 mg/m² and for docetaxel in about 75 to 150 mg/m² percourse of treatment. These dosage may be administered for example once,twice or more per course of treatment, which may be repeated for exampleevery 7,14, 21 or 28 days.

5. Platinum Compounds

In another embodiment of the invention, the cytotoxic compound is aplatinum compound.

Definition: The term “platinum compounds” as used herein refers to anytumour cell growth inhibiting platinum compound including platinumcoordination compounds, compounds which provide platinum in the form ofan ion and analogues of platinum compounds as described herein,including the ionic, salt, solvate, isomers, tautomers, N-oxides, ester,prodrugs, isotopes and protected forms thereof (preferably the salts ortautomers or isomers or N-oxides or solvates thereof, and morepreferably, the salts or tautomers or N-oxides or solvates thereof), asdescribed above.

Technical background: In the chemotherapeutic treatment of cancers,cisplatin (cis-diaminodichloroplatinum (II)) has been used successfullyfor many years in the treatment of various human solid malignant tumoursfor example testicular cancer, ovarian cancer and cancers of the headand neck, bladder, oesophagus and lung.

More recently, other diamino -platinum complexes, for examplecarboplatin (diamino(1,1-cyclobutane-dicarboxylato)platinum (II)), havealso shown efficacy as chemotherapeutic agents in the treatment ofvarious human solid malignant tumours, carboplatin being approved forthe treatment of ovarian cancer. A further antitumour platinum compoundis oxaliplatin (L-OHP), a third generation diamino-cyclohexaneplatinum-based cytotoxic drug, which has the chemical name(1,2-diaminocyclohexane)oxalato-platinum (II). Oxaliplatin is used, forexample, for the treatment of metastatic colorectal cancer, based on itslack of renal toxicity and higher efficacy in preclinical models ofcancer in comparison to cisplatin.

Biological activity: The platinum compounds of the combinations of theinvention have activity against various cancers., in particular againsta sub-set of cancers including solid malignant tumours (for exampletesticular cancer), ovarian cancer, metastatic colorectal cancer andcancers of the head and neck, bladder, oesophagus and lung.

Problems: Although cisplatin and other platinum compounds have beenwidely used as chemotherapeutic agents in humans, they are nottherapeutically effective in all patients or against all types oftumours. Moreover, such compounds need to be administered at relativelyhigh dosage levels which can lead to toxicity problems such as kidneydamage. Also, and especially with cisplatin, the compounds cause nauseaand vomiting in patients to a varying extent, as well as leucopenia,anemia and thrombocytopenia. There is therefore a need to increaseefficacy and also to provide a means for the use of lower dosages toreduce the potential of adverse toxic side effects to the patient.

Preferences: Preferred platinum compounds for use in accordance with theinvention include cisplatin, carboplatin and oxaliplatin. Other platinumcompounds include chloro(diethylenediamino)-platinum (II) chloride;dichloro(ethylenediamino)-platinum (II); spiroplatin; iproplatin;diamino(2-ethylmalonato)platinum (II);(1,2-diaminocyclohexane)malonatoplatinum (II);(4-carboxyphthalo)-(1,2-diaminocyclohexane)platinum (II);(1,2-diaminocyclohexane)-(isocitrato)platinum (II);(1,2-diaminocyclohexane)-cis-(pyruvato)platinum (II); onnaplatin; andtetraplatin. Cisplatin is commercially available for example under thetrade name Platinol from Bristol-Myers Squibb Corporation as a powderfor constitution with water, sterile saline or other suitable vehicle.Cisplatin may also be prepared for example as described by G. B.Kauffman and D. O. Cowan, Inorg. Synth. 7, 239 (1963), or by processesanalogous thereto. Carboplatin is commercially available for examplefrom Bristol-Myers Squibb Corporation under the trade name Paraplatin,or may be prepared for example as described in U.S. Pat. No. 4,140,707,or by processes analogous thereto. Oxaliplatin is commercially availablefor example from Sanofi-Synthelabo Inc under the trade name Eloxatin, ormay be prepared for example as described in U.S. Pat. No. 4,169,846, orby processes analogous thereto. Other platinum compounds and theirpharmaceutical compositions are commercially available and/or can beprepared by conventional techniques.

Specific embodiments: In one embodiment, the platinum compound isselected from chloro(diethylenediamino)-platinum (II) chloride;dichloro(ethylenediamino)-platinum (II); spiroplatin; iproplatin;diamino(2-ethylmalonato)platinum (II);(1,2-diaminocyclohexane)malonatoplatinum (II);(4-carboxyphthalo)-(1,2-diaminocyclohexane)platinum (II);(1,2-diaminocyclohexane)-(isocitrato)platinum (II);(1,2-diaminocyclohexane)-cis-(pyruvato)platinum (II); onnaplatin;tetraplatin, cisplatin, carboplatin and oxaliplatin. In anotherembodiment, the platinum compound is a platinum compound other thancisplatin, for example a platinum compound such aschloro(diethylenediamino)-platinum (II) chloride;dichloro(ethylenediamino)-platinum (II); spiroplatin; iproplatin;diamino(2-ethylmalonato)platinum (II);(1,2-diaminocyclohexane)malonatoplatinum (II);(4-carboxyphthalo)-(1,2-diaminocyclohexane)platinum (II);(1,2-diaminocyclohexane)-(isocitrato)platinum (II);(1,2-diaminocyclohexane)-cis-(pyruvato)platinum (II); onnaplatin;tetraplatin, carboplatin or oxaliplatin, preferably selected fromcarboplatin and oxaliplatin.

Posology: The platinum coordination compound is advantageouslyadministered in a dosage of 1 to 500 mg per square meter (mg/m²) of bodysurface area, for example 50 to 400 mg/m² particularly for cisplatin ina dosage of about 75 mg/m², for carboplatin in about 300 mg/m² and foroxaliplatin in about 50-100 mg/m². These dosages may be administered forexample once, twice or more per course of treatment, which may berepeated for example every 7, 14, 21 or 28 days.

6. Topoisomerase 2 Inhibitors

In another embodiment of the invention, the cytotoxic compound is atopoisomerase 2 inhibitor.

Definition: The term “topoisomerase 2 inhibitor” as used herein refersto topoisomerase 2 inhibitor or analogues of topoisomerase 2 inhibitoras described above, including the ionic, salt, solvate, isomers,tautomers, N-oxides, ester, prodrugs, isotopes and protected formsthereof (preferably the salts or tautomers or isomers or N-oxides orsolvates thereof, and more preferably, the salts or tautomers orN-oxides or solvates thereof), as described above.

Technical background: An important class of anticancer drugs are theinhibitors of the enzyme topoisomerase 2 which causes double-strandbreaks to release stress build-up during DNA transcription andtranslation. Compounds that inhibit the function of this enzyme aretherefore cytotoxic and useful as anti-cancer agents.

Among the topoisomerase 2 inhibitors which have been developed and usedin cancer chemotherapy are the podophyllotoxins. These drugs act by amechanism of action which involves the induction of DNA strand breaks byan interaction with DNA topoisomerase 2 or the formation of freeradicals. Podophyllotoxin, which is extracted from the mandrake plant,is the parent compound from which two glycosides have been developedwhich show significant therapeutic activity in several human neoplasms,including pediatric leukemia, small cell carcinomas of the lung,testicular tumours, Hodgkin's disease, and large cell lymphomas. Thesederivatives are etoposide (VP-16), which has the chemical name4′-demethylepipodophyllotoxin9-[4,6-O—(R)-ethylidene-β-D-glucopyranoside], and teniposide (VM-26),which has the chemical name 4′-demethylepipodophyllotoxin9-[4,6-0-(R)-2-thenylidene-β-D-glucopyranoside].

Both etoposide and teniposide, however, suffer from certain toxicside-effects especially myelosuppression. Another important class oftopoisomerase 2 inhibitors are the anthracycline derivatives which areimportant anti-tumour agents and comprise antibiotics obtained from thefungus Streptomyces peuticus var. caesius and their derivatives,characterized by having a tetracycline ring structure with an unusualsugar, daunosamine, attached by a glycosidic linkage. Among thesecompounds, the most widely used include daunorubicin, which has thechemical name7-(3-amino-2,3,6-trideoxy-L-lyxohexosyloxy)-9-acetyl-7,8,9,10-tetrahydro-6,9,11-trihydroxy-4-methoxy-5,12-naphthacenequinone,doxorubicin, which has the chemical name10-[(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxylacetyl)-I-methoxy-5,12-naphthacenedione,and idarubicin, which has the chemical name9-acetyl-[(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11-trihydroxy-5,12-naphthacenedione.

Daunorubicin and idarubicin have been used primarily for the treatmentof acute leukaemias whereas doxorubicin displays broader activityagainst human neoplasms, including a variety of solid tumoursparticularly breast cancer. Another anthracycline derivatives which isuseful in cancer chemotherapy is epirubicin. Epirubicin, which has thechemical name(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-arabino-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione,is a doxorubicin analog having a catabolic pathway that involvesglucuronidation, by uridine diphosphate-glucuronosyl transferase in theliver (unlike that for doxorubicin), which is believed to account forits shorter half-life and reduced cardiotoxicity. The compound has beenused for the treatment of various cancers including cervical cancer,endometrial cancer, advanced breast cancer and carcinoma of the bladderbut suffers from the side-effects of myelosuppression andcardiotoxicity. The latter side-effect is typical of anthracyclinederivatives which generally display a serious cardiomyopathy at higherdoses, which limits the doses at which these compounds can beadministered. A further type of topoisomerase 2 inhibitor is representedby mitoxantrone, which has the chemical name1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione,and is used for the treatment of multiple sclerosis, non-Hodgkin'slymphoma, acute myelogenous leukaemia, and breast, prostate and livertumours. Others include losoxantrone and actinomycin D.

Side-effects from administration of mitoxantrone includemyelosuppression, nausea, vomiting, stomatitis, alopecia but lesscardiotoxicity than anthracyclines.

Biological activity: The topoisomerase 2 inhibitors of the combinationsof the invention have activity against various cancers as describedabove. In particular, they have activity against a sub-set of cancersincluding leukemia (e.g. acute leukaemias), small cell carcinomas of thelung, testicular tumours, Hodgkin's disease, large cell lymphomas,breast cancer, cervical cancer, endometrial cancer, advanced breastcancer and carcinoma of the bladder.

Problems: This class of cytotoxic compound is associated with sideeffects, as mentioned above. Thus, there is a need to provide a meansfor the use of lower dosages to reduce the potential of adverse toxicside effects to the patient.

Preferences: Preferred topoisomerase 2 inhibitor compounds for use inaccordance with the invention include anthracycline derivatives,mitoxantrone and podophyllotoxin derivatives as defined to herein.

Preferred anti-tumour anthracycline derivatives for use in accordancewith the invention include daunorubicin, doxorubicin, idarubicin andepirubicin referred to above. Daunorubicin is commercially available forexample as the hydrochloride salt from Bedford Laboratories under thetrade name Cerubidine, or may be prepared for example as described inU.S. Pat. No. 4,020,270, or by processes analogous thereto. Doxorubicinis commercially available for example from Pharmacia and Upjohn Co underthe trade name Adriamycin, or may be prepared for example as describedin U.S. Pat. No. 3,803,124, or by processes analogous thereto.Doxorubicin derivatives include pegylated doxorubicin hydrochloride andliposome-encapsulated doxorubicin citrate. Pegylated doxorubicinhydrochloride is commercially available from Schering-PloughPharmaceuticals under the trade name Caeylx; liposome-encapsulateddoxorubicin citrate is commercially available for example from ElanCorporation under the trade name Myocet. Idarubicin is commerciallyavailable for example as the hydrochloride salt from Pharmacia & Upjohnunder the trade name Idamycin, or may be prepared for example asdescribed in U.S. Pat. No. 4,046,878, or by processes analogous thereto.Epirubicin is commercially available for example from Pharmacia andUpjohn Co under the trade name Pharmorubicin, or may be prepared forexample as described in U.S. Pat. No 4,058,519, or by processesanalogous thereto. Mitoxantrone is commercially available for example ofOSI Pharmaceuticals, under the trade name Novantrone, or may be preparedfor example as described in U.S. Pat. No. 4,197,249, or by processesanalogous thereto.

Other anti-tumour anthracycline derivatives may be prepared inconventional manner for example by processes analogous to thosedescribed above for the specific anthracycline derivatives.

Preferred anti-tumour podophyllotoxin derivatives for use in accordancewith the invention include etoposide and teniposide referred to above.Etoposide is commercially available for example from Bristol-MyersSquibb Co under the trade name VePesid, or may be prepared for exampleas described in European patent specification No111058, or by processesanalogous thereto. Teniposide is commercially available for example fromBristol-Myers Squibb Co under the trade name Vumon, or may be preparedfor example as described in PCT patent specification No. WO 93/02094, orby processes analogous thereto. Other anti-tumour podophyllotoxinderivatives may be prepared in conventional manner for example byprocesses analogous to those described above for etoposide andteniposide.

Specific embodiments: In one embodiment, the topoisomerase 2 inhibitoris an anthracycline derivative, mitoxantrone or a podophyllotoxinderivative. In another embodiment, the topoisomerase 2 inhibitor isselected from daunorubicin, doxorubicin, idarubicin and epirubicin. In afurther embodiment, the topoisomerase 2 inhibitor is selected frometoposide and teniposide. Thus, in a preferred embodiment, thetopoisomerase 2 inhibitor is etoposide. In another embodiment, thetopoisomerase 2 inhibitor is an anthracycline derivative other thandoxorubicin, for example a topoisomerase 2 inhibitor such asdaunorubicin, idarubicin and epirubicin.

Posology: The anti-tumour anthracycline derivative is advantageouslyadministered in a dosage of 10 to 150 mg per square meter (mg/m²) ofbody surface area, for example 15 to 60 mg/m², particularly fordoxorubicin in a dosage of about 40 to 75 mg/m², for daunorubicin in adosage of about 25 to 45mg/m², for idarubicin in a dosage of about 10 to15 mg/m² and for epirubicin in a dosage of about 100-120 mg/m².

Mitoxantrone is advantageously administered in a dosage of about 12 to14 mg/m² as a short intravenous infusion about every 21 days.

The anti-tumour podophyllotoxin derivative is advantageouslyadministered in a dosage of 30 to 300 mg/m² of body surface area, forexample 50 to 250 mg/m particularly for etoposide in a dosage of about35 to 100 mg/m, and for teniposide in about 50 to 250 mg/m².

The dosages noted above may generally be administered for example once,twice or more per course of treatment, which may be repeated for exampleevery 7,14, 21 or 28 days.

The antibiotic bleomycin may also be used as a cytotoxic agent as anancillary compound according to the invention.

7. Signalling Inhibitors

In another embodiment of the invention, the combination comprises asignaling inhibitor.

Definition: The term “signalling inhibitor” as used herein refers tosignalling inhibitors or analogues of signalling inhibitors as describedherein, including the ionic, salt, solvate, isomers, tautomers,N-oxides, ester, prodrugs, isotopes and protected forms thereof(preferably the salts or tautomers or isomers or N-oxides or solvatesthereof, and more preferably, the salts or tautomers or N-oxides orsolvates thereof), as described above.

Technical background: A malignant tumour is the product of uncontrolledcell proliferation. Cell growth is controlled by a delicate balancebetween growth-promoting and growth-inhibiting factors. In normal tissuethe production and activity of these factors results in differentiatedcells growing in a controlled and regulated manner that maintains thenormal integrity and functioning of the organ. The malignant cell hasevaded this control; the natural balance is disturbed (via a variety ofmechanisms) and unregulated, aberrant cell growth occurs.

One driver for growth is the epidermal growth factor (EGF), and thereceptor for EGF (EGFR) has been implicated in the development andprogression of a number of human solid tumours including those of thelung, breast, prostate, colon, ovary, head and neck. EGFR is a member ofa family of four receptors, namely EGFR (HER1 or ErbB1), ErbB2(HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). These receptors are largeproteins that reside in the cell membrane, each having a specificexternal ligand binding domain, a transmembrane domain and an internaldomain which has tyrosine kinase enzyme activity. When EGF attaches toEGFR, it activates the tyrosine kinase, triggering reactions that causethe cells to grow and multiply. EGFR is found at abnormally high levelson the surface of many types of cancer cells, which may divideexcessively in the presence of EGF. Inhibition of EGFR activity hastherefore been a target for chemotherapeutic research in the treatmentof cancer. Such inhibition can be effected by direct interference withthe target EGFR on the cell surface, for example by the use ofantibodies, or by inhibiting the tyrosine kinase activity associatedwith the activated receptor.

Examples of antibodies which target EGFR are the monoclonal antibodiestrastuzumab and cetuximab. Amplification of the human epidermal growthfactor receptor 2 protein (HER 2) in primary breast carcinomas has beenshown to correlate with a poor clinical prognosis for certain patients.Trastuzumab is a highly purified recombinant DNA-derived humanizedmonoclonal IgG1 kappa antibody that binds with high affinity andspecificity to the extracellular domain of the HER2 receptor. In vitroand in vivo preclinical studies have shown that administration oftrastuzumab alone or in combination with paclitaxel or carboplatinsignificantly inhibits the growth of breast tumour-derived cell linesthat over-express the HER2 gene product. In clinical studies trastuzumabhas been shown to have clinical activity in the treatment of breastcancer. The most common adverse effects of trastuzumab are fever andchills, pain, asthenia, nausea, vomiting, diarrhea, headache, dyspnea,rhinitis, and insomnia. Trastuzumab has been approved for the treatmentof metastatic breast cancer involving over-expression of the HER2protein in patients who have received one or more chemotherapy regimes.

Cetuximab has been used for the treatment of irotecan-refractorycolorectal cancer. It is also being evaluated both as a single agent andin combination with other agents for use in the treatment of a varietyof other cancers for example head and neck cancer, metastatic pancreaticcarcinoma, and non-small-cell lung cancer. The administration ofcetuximab can cause serious side effects, which may include difficultyin breathing and low blood pressure.

Examples of agents which target EGFR tyrosine kinase activity includethe tyrosine kinase inhibitors gefitinib and erlotinib. Gefitinib whichhas the chemical name4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,is used for the treatment of non-small-cell lung cancer, and is alsounder development for other solid tumours that over-express EGFreceptors such as breast and colorectal cancer. It has been found thatpatients receiving gefitinib may develop interstitial lung disease thatcauses inflammation within the lung. Eye irritation has also beenobserved in patients receiving gefitinib. Erlotinib, which has thechemical nameN-(3-ethynyl-phenyl)-6,7-bis(2-methoxyethoxy)-4-quinazoline, has alsobeen used for the treatment of non-small-cell lung cancer, and is beingdeveloped for the treatment of various other solid tumours such aspancreatic cancer, the most common side effects being rash, loss ofappetite and fatigue; a more serious side effect which has been reportedis interstitial lung disease.

Another growth factor which has received attention as a target foranticancer research is the vascular endothelial growth factor (VEGF).VEGF acts via association with a family of cell surface receptors and isa key regulator of vasculogenesis during angiogenic processes includingwound healing, retinopathy, psoriasis, inflammatory disorders, tumourgrowth and metastasis. Studies have shown that over-expression of VEGFis strongly associated with invasion and metastasis in human malignantdisease.

An example of an antibody that targets the VEGF/VEGF receptor system isthe monoclonal antibody bevacizumab which is a recombinant humanisedmonoclonal IgG1 antibody that binds to and inhibits the growth factorVEGF. Bevacizumab has been used for the treatment of colorectal cancer,for example in combination with 5-fluorouracil. Bevacizumab is alsobeing developed as a potential treatment for other solid tumours such asmetastatic breast cancer, metastatic non-small-cell lung cancer andrenal cell carcinoma. The most serious adverse events associated withbevacizumab include gastrointestinal perforations, hypertensive crises,nephrotic syndrome and congestive heart failure. Other therapeuticagents in development which target the action of VEGF at alternatepoints in the signal transduction cascade intiated by this growth factorinclude sunitinib which is marketed under the trade name Sutent bySugen/Pfizer and inhibits the kinase activity of the VEGF receptor.Sutent has demonstrated efficacy in Phase III trials in gastrointestinaltumours.

Another growth factor of importance in tumour development is theplatelet-derived growth factor (PDGF) that comprises a family of peptidegrowth factors that signal through cell surface tyrosine kinasereceptors (PDGFR) and stimulate various cellular functions includinggrowth, proliferation, and differentiation. PDGF expression has beendemonstrated in a number of different solid tumours includingglioblastomas and prostate carcinomas. The tyrosine kinase inhibitorimatinib mesylate, which has the chemical name4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-ylpyridinyl]amino]-phenyl]benzamidemethanesulfonate, blocks activity of the Bcr-Abl oncoprotein and thecell surface tyrosine kinase receptor c-Kit, and as such is approved forthe treatment on chronic myeloid leukemia and gastrointestinal stromaltumours. Imatinib mesylate is also a potent inhibitor of PDGFR kinaseand is currently being evaluated for the treatment of chronicmyelomonocytic leukemia and glioblastoma multiforme, based upon evidencein these diseases of activating mutations in PDGFR. The most frequentlyreported drug-related adverse events were edema, nausea, vomiting,cramps and musculosketetal pain.

A further growth factor target for cancer chemotherapy is inhibition ofRaf which is a key enzyme in the signal transduction pathway thattriggers cell growth. Abnormal activation of this pathway is a commonfactor in the development of most cancers, including two-thirds ofmelanomas. By blocking the action of Raf kinase, it may be possible toreverse the progression of these tumours. One such inhibitor issorafenib (BAY 43-9006) which has the chemical name4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-N2-methylpyridine-2-carboxamide.Sorafenib targets both the Raf signalling pathway to inhibit cellproliferation and the VEGFR/PDGFR signalling cascades to inhibit tumourangiogenesis. Raf kinase is a specific enzyme in the Ras pathway.Mutations in the Ras gene occur in approximately 20 percent of all humancancers, including 90 percent of pancreatic cancers, 50 percent of coloncancers and 30 percent of non-small cell lung cancers. Sorafenib isbeing investigated for the treatment of a number of cancers includingliver and kidney cancer. The most common side effects of sorafenib arepain, swelling, redness of the hands and/or feet, and also rash, fatigueand diarrhea.

Biological activity: The signalling inhibitors of the combinations ofthe invention are specific inhibitors of cell signalling proteins asdescribed above and have activity against various cancers. Combinationsof compounds of Formula I with signalling inhibitors may be beneficialin the treatment and diagnosis of many types of cancer. Combination witha molecularly targeted agent such as a signalling inhibitor (e.g.Iressa, Avastin, herceptin, or Gleevec™) would find particularapplication in relation to cancers which express or have activated therelevant molecular target such as EGF receptor, VEGF receptor, ErbB2,BCRabl, c-kit, PDGF. Diagnosis of such tumours could be performed usingtechniques known to a person skilled in the art and as described hereinsuch as RTPCR and FISH.

Problems: There is a need to increase the inhibitory efficacy ofsignalling inhibitors against tumour growth and also to provide a meansfor the use of lower dosages of signaling inhibitors to reduce thepotential for adverse toxic side effects to the patient.

Preferences: Preferred signalling inhibitors for use in accordance withthe invention include antibodies targeting EGFR such as monoclonalantibodies trastuzumab and cetuximab, EGFR tyrosine kinase inhibitorssuch as gefitinib and erlotinib, VEGF targeting antibody is bevacizumab,PDGFR inhibitor such as imatinib mesylate and Raf inhibitor such assorafenib referred to herein.

Preferred antibodies targeting EGFR include the monoclonal antibodiestrastuzumab and cetuximab. Trastuzumab is commercially available fromGenentech Inc under the trade name Herceptin, or may be obtained asdescribed in U.S. Pat. No. 5,821,337. Cetuximab is commerciallyavailable from Bristol-Myers Squibb Corporation under the trade nameErbitux, or may be obtained as described in PCT patent specification No.WO 96/40210.

Preferred EGFR tyrosine kinase inhibitors include gefitinib anderlotinib. Gefitinib is commercially available from AstraZeneca plcunder the trade name Iressa, or may be obtained as described in PCTpatent specification No. WO 96/33980. Erlotinib is commerciallyavailable from Pfizer Inc under the trade name Tarceva, or may beobtained as described in PCT patent specification No. WO 96/30347.

A preferred antibody targeting VEGF is bevacizumab which is commerciallyavailable from Genentech Inc under the trade name Avastin, or may beobtained as described in PCT patent specification No. WO 94/10202.

A preferred PDGFR inhibitor is imatinib mesylate which is commerciallyavailable from Novartis AG under the trade name Gleevec™ (a.k.a.Glivec®), or may be obtained as described in European patentspecification No 564409.

A preferred Raf inhibitor is sorafenib which is available from Bayer AG,or may be obtained as described in PCT patent specification No. WO00/42012.

Specific embodiments: In one embodiment, the signalling inhibitor isgefitinib (Iressa). In other embodiments the signalling inhibitor isselected from trastuzumab, cetuximab, gefitinib, erlotinib, bevacizumab,imatinib mesylate and sorafenib.

Posology: With regard to the EGFR antibodies, these are generallyadministered in a dosage of 1 to 500 mg per square meter (mg/m²) of bodysurface area, trastuzumab being advantageously administered in a dosageof 1 to 5 mg/m² of body surface area, particularly 2 to 4 mg/m²;cetuxumab is advantageously administered in a dosage of about 200 to 400mg/m², preferably about 250 mg/m².

With regard to the EGFR tyrosine kinase inhibitors, these are generallyadministered in a daily oral dosage of 100 to 500 mg, for examplegefitinib in a dosage of about 250 mg and erlotinib in a dosage of about150 mg.

With regard to the VEGF monoclonal antibody bevacizumab, this isgenerally administered in a dosage of about 1 to 10 mg/kg for exampleabout 5 mg/kg.

With regard to the PDGF inhibitor imatinib, this is generallyadministered in a dosage of about 400 to 800 mg per day preferably about400 mg per day.

With regard to the Raf inhibitor sorfenib, this is still underevaluation but a possible dosage is about 800 mg daily.

These dosages may be administered for example once, twice or more percourse of treatment, which may be repeated for example every 7, 14, 21or 28 days.

PKB Pathway Inhibitors

Another preferred class of signaling inhibitor for use in thecombinations of the invention are PKB pathway inhibitors. PKB pathwayinhibitors are those that inhibit the activation of PKB, the activity ofthe kinase itself or modulate downstream targets, blocking theproliferative and cell survival effects of the pathway. Target enzymesin the pathway include Phosphatidyl inositol-3 kinase (PI3K), PKBitself, Mammalian target of rapamycin (MTOR), PDK-1 and p70 S6 kinaseand forkhead translocation.

Several components of the PI 3-kinase/PKB/PTEN pathway are implicated inoncogenesis. In addition to growth factor receptor tyrosine kinases,integrin-dependent cell adhesion and G-protein coupled receptorsactivate PI 3-kinase both directly and indirectly through adaptormolecules. Functional loss of PTEN (the most commonly mutatedtumour-suppressor gene in cancer after p53), oncogenic mutations in PI3-kinase, amplification of PI 3-kinase and overexpression of PKB havebeen established in many malignancies. In addition, persistent signalingthrough the PI 3-kinase/PKB pathway by stimulation of the insulin-likegrowth factor receptor is a mechanism of resistance to epidermal growthfactor receptor inhibitors.

The discovery of non-random, somatic mutations in the gene encodingp110α in a range of human tumours suggests an oncogenic role for themutated PI 3-kinase enzyme (Samuels, et al., Science, 304 554, April2004). Mutations in p110α have since been detected in the followinghuman tumours: colon (32%), hepatocellular (36%) and endometroid andclear cell cancer (20%). p110α is now the most commonly mutated gene inbreast tumours (25-40%). Forkhead family translocations often occur inacute leukemia.

The PI 3-kinase/PKB/PTEN pathway is thus an attractive target for cancerdrug development since such agents would be expected to inhibitproliferation and surmount resistance to cytotoxic agents in cancercells. Examples of PKB pathway inhibitors include PI3K Inhibitors suchas Semaphore, SF1126 and MTOR inhibitors such as Rapamycin Analogues.RAD 001 (everolimus) from Novartis is an orally available derivative ofthe compound rapamycin. The compound is a novel macrolide, which isbeing developed as an antiproliferative drug with applications as animmunosuppressant and anticancer agent. RAD001 exerts its activity ongrowth-factor dependent proliferation of cells through its high affinityfor an intracellular receptor protein, FKBP-12. The resultingFKBP-12/RAD001 complex then binds with mTOR to inhibit downstreamsignaling events. The compound is currently in clinical development fora wide variety of oncology indications. CCI 779 (temsirolemus) fromWyeth Pharmaceuticals and AP23573 from Ariad Pharmaceuticals are alsorapamycin analogues. AP23841 and AP23573 from Ariad Pharmaceutical alsotarget mTOR. Calmodulin inhibitors from Harvard are forkheadtranslocation inhibitors. (Nature Reviews drug discovery, Exploiting thePI3K/AKT Pathway for Cancer Drug Discovery; Bryan T. Hennessy, Debra L.Smith, Prahlad T. Ram, Yiling Lu and Gordon B. Mills; December 2005,Volume 4; pages 988-1004).

Preferred PKB pathway inhibitors for use in the combinations of theinvention include PKB inhibitors, as described in more detail below:

Definition: The term “PKB inhibitor” is used herein to define a compoundwhich inhibits or modulates protein kinase B (PKB), including the ionic,salt, solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopesand protected forms thereof (preferably the salts or tautomers orisomers or N-oxides or solvates thereof, and more preferably, the saltsor tautomers or N-oxides or solvates thereof), as described above.

Technical background: KRX-0401 (Perifosine/NSC 639966) is a syntheticsubstituted heterocyclic alkylphosphocholine that acts primarily at thecell membrane targeting signal transduction pathways, includinginhibition of PKB phosphorylation. KRX-0401 has been evaluated in phase1 studies as a potential oral anticancer drug. Dose limiting toxicitiesincluded nausea, vomiting and fatigue. Gastrointestinal toxicitiesincreased at higher doses. A phase II trial in refractory sarcoma isplanned.

API-2/TCN is a small molecule inhibitor of PKB signaling pathway intumour cells. Phase I and II clinical trials of API-2/TCN have beenconducted on advanced tumours. API-2/TCN exhibited some side effects,which include hepatotoxicity, hypertriglyceridemia, thrombocytopenia,and hyperglycemia. Due to its severe side effects at high doses,API-2/TCN has been limited in the clinic.

RX-0201 is being developed as an AKT protein kinase inhibitor for thetreatment of solid tumours. In July 2004, a phase I trial was initiatedin patients with advanced or metastasized cancers. Data from this showedRX-0201 inhibited overexpression of Akt and suppressed cancer growth inbrain, breast, cervix, liver, lung, ovary, prostate and stomach tumours,and was well tolerated. By March 2005, US Orphan Drug status had beengranted to RX-0201 for several solid tumour types.

Enzastaurin HCl (LY317615) suppresses angiogenesis and was advanced forclinical development based upon anti-angiogenic activity. It isdescribed as a selective PKCβ inhibitor. It also has a directanti-tumour effect, and suppresses GSK3β phosphorylation.

SR-13668 is claimed to be an orally active specific AKT inhibitor thatsignificantly inhibits phospho-AKT in breast cancer cells both in vitroand in vivo. In vivo assessment in mice showed no adverse effects atdoses 10 times more than were needed for antitumour activity.

PX-316 is a D-3-deoxy-phosphatidyl-myo-inositol that binds to the PHdomain of PKB, trapping it in the cytoplasm and thus preventing PKBactivation. Anti-tumour activity was seen in early xenografts and waswell tolerated.

Allosteric, selective inhibitors of PKB based on a2,3-diphenylquinoxaline core or a 5,6-diphenylpyrazin-2(1 H)-one corehave been developed (Merck).

KRX-0401: In a Phase I weekly dosing study conducted in Europe, therecommended Phase II dose was 600/mg/week. Subsequent studies conductedin the U.S. have shown that much higher doses are well tolerated whenthe doses are divided and administered at 4 to 6 hour intervals. Inaddition, it has been shown that KRX-0401 has a very long half-life inthe range of 100 hours. This makes the possibility of a relativenon-toxic, intermittent dosing schedule very plausible.

A phase I trial of API-2 was conducted using a 5-day continuous infusionschedule. Dose levels ranged from 10 mg/sq m/day×5 days to 40 mg/sqm/day×5 days. Initially, courses were repeated every 3 to 4 weeks. Ascumulative toxicity became manifested, the interval between courses waschanged to every 6 weeks. Recommended schedule for Phase II studies is20 mg/sq m/day for 5 days every 6 weeks. A Phase II trial of TCN-P wasconducted in metastatic or recurrent squamous cell carcinoma of thecervix using a 5-day continuous infusion schedule. The starting dose was35 mg/m^(2×5) days and courses were repeated every 6 weeks.

Further PKB inhibitors include Perifosine from Keryx Biopharmaceuticals.Perifosine is an oral Akt inhibitor which exerts a marked cytotoxiceffect on human tumour cell lines, and is currently being tested inseveral phase II trials for treatment of major human cancers. KRX-0401(Perifosine/NSC 639966) has the structure:

It can be prepared according to Aste Medica patent publication DE4222910or Xenoport patent publication US2003171303.

API-2/TCN (Triciribine) has the structure:

It can be prepared according to Bodor patent publication WO9200988 orRibapharm patent publication WO2003061385.

Enzastaurin hydrochloride has the structure:

It can be prepared according to Eli Lilly patent publicationWO2004006928.

SR 13668 has the structure:

It can be prepared according to SRI International patent publicationUS2004043965.

NL-71-101 has the structure:

It can be prepared according to Biochemistry (2002), 41(32), 10304-10314or Peptor patent publication WO2001091754.

DeveloGen (formerly Peptor) is investigating NL-71-101, a protein kinaseB (PKB) inhibitor, for the potential treatment of cancer [466579],[539004]. At the beginning of 2003, the compound was undergoing leadoptimization [495463]. By February 2004, the company was seeking tooutlicense certain development rights to its protein kinase B program[523638].

In 2002, data were published showing that NL-71-101 inhibited theactivity of PKB over PKA, PKG and PKC with IC50 values of 3.7, 9, 36 and104 microM, respectively. NL-71-101 induced apoptosis in OVCAR-3 tumourcells, in which PKB is amplified at concentrations of 50 and 100 microM[466579]. This compound has the structure:

Specific embodiments: Embodiments contemplated include combinations inwhich the anti-cancer agent is a PKB inhibitor selected from one or moreof the specific compounds described above.

Pharmaceutical Formulations

While it is possible for the active compounds in the combinations of theinvention to be administered without any accompanying pharmaceuticalexcipients or carriers, it is preferable to present them in the form ofpharmaceutical compositions (e.g. formulations). As such, they may beformulated for simultaneous or sequential administration.

Where they are intended for sequential administration, they willtypically be formulated in separate compositions which may be of thesame type or a different type. Thus, for example, the components of thecombination may be formulated for delivery by the same route (e.g. bothby the oral route or both by injection) or they may be formulated foradministration by different routes (e.g. one by the oral route andanother by a parenteral route such as by i.v. injection or infusion). Ina preferred embodiment the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide and salts thereof, particularly acid addition saltssuch as the methanesulphonic acid, acetic acid and hydrochloric acidsalts is administered sequentially (either before or after) orsimulatenously with the ancillary compound. Preferably the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide and salts thereof, particularly acid addition saltssuch as the methanesulphonic acid, acetic acid and hydrochloric acidsalts is administered using an i.v. formulation as defined herein.

When they are intended for simultaneous administration, they may beformulated together or separately and, as above, may be formulated foradministration by the same route or by different routes.

The compositions typically comprise at least one active compound of thecombination together with one or more pharmaceutically acceptablecarriers, adjuvants, excipients, diluents, fillers, buffers,stabilisers, preservatives, lubricants, or other materials well known tothose skilled in the art. The compositions may also include othertherapeutic or prophylactic agents, for example agents that reduce oralleviate some of the side effects associated with chemotherapy.Particular examples of such agents include anti-emetic agents and agentsthat prevent or decrease the duration of chemotherapy-associatedneutropenia and prevent complications that arise from reduced levels ofred blood cells or white blood cells, for example erythropoietin (EPO),granulocyte macrophage-colony stimulating factor (GM-CSF), andgranulocyte-colony stimulating factor (G-CSF).

Also included are agents that inhibit bone resorption such asbisphosphonate agents e.g. zoledronate, pamidronate and ibandronate, aswell as agents that suppress inflammatory responses (such asdexamethazone, prednisone, and prednisolone). Also included are agentsused to reduce blood levels of growth hormone and IGF-I in acromegalypatients such as synthetic forms of the brain hormone somatostatin,which includes octreotide acetate which is a long-acting octapeptidewith pharmacologic properties mimicking those of the natural hormonesomatostatin. Further included are agents such as leucovorin, which isused as an antidote to drugs that decrease levels of folic acid, orfolinic acid it self. In one particular embodiment is the combination of5FU and leucovorin or 5FU and folinic acid. In addition megestrolacetate can be used for the treatment of side-effects including oedemaand thromoembolic episodes.

Therefore in one embodiment the combinations further include anadditional agent selected from erythropoietin (EPO), granulocytemacrophage-colony stimulating factor (GM-CSF), granulocyte-colonystimulating factor (G-CSF), zoledronate, pamidronate, ibandronate,dexamethazone, prednisone, prednisolone, leucovorin, folinic acid andmegestrol acetate.

In particular the combinations further include an additional agentselected from erythropoietin (EPO), granulocyte macrophage-colonystimulating factor (GM-CSF), granulocyte-colony stimulating factor(G-CSF), zoledronate, pamidronate, dexamethazone, prednisone,prednisolone, leucovorin, and folinic acid such as erythropoietin (EPO),granulocyte macrophage-colony stimulating factor (GM-CSF) andgranulocyte-colony stimulating factor (G-CSF).

Zoledronic acid is available from Novartis under the Tradename Zometa®.It is used in the treatment of bone metastasis in a variety of tumortypes and for the treatment of hypercalcemia.

Pamidronate disodium (APD) available from Novartis under the tradenameAredia is a bone-resorption inhibitor and is used in the treatment ofmoderate or severe hypercalcemia. Pamidronate disodium is for i.v.injection.

Octreotide acetate is available from Novartis as Sandostatin LAR®(octreotide acetate for injectable suspension) and Sandostatin®(octreotide acetate for injection ampuls or for vials). Octreotide isknown chemically as L-Cysteinamide,D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxy-methyl)propyl]-,cyclic (2, 7)-disulfide; [R—(R*,R*)]. Synthetic forms of the brainhormone somatostatin, such as octreotide, work at the site of thetumour. They bind to sst-2/sst-5 receptors to regulate gastrointestinalhormone secretion and affect tumour growth.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one active compound, as definedabove, together with one or more pharmaceutically acceptable carriers,excipients, buffers, adjuvants, stabilizers, or other materials, asdescribed herein.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of a subject (e.g. human) without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. Each carrier,excipient, etc. must also be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation.

Accordingly, in a further aspect, the invention provides combinations ofa cytotoxic compound or signalling inhibitor and a compound of theformula (0) or a sub-group thereof such as formulae (I⁰), (I), (Ia),(Ib), (II), (III) (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII)and sub-groups thereof as defined herein in the form of pharmaceuticalcompositions.

The pharmaceutical compositions can be in any form suitable for oral,parenteral, topical, intranasal, ophthalmic, otic, rectal,intra-vaginal, or transdermal administration. Where the compositions areintended for parenteral administration, they can be formulated forintravenous, intramuscular, intraperitoneal, subcutaneous administrationor for direct delivery into a target organ or tissue by injection,infusion or other means of delivery. The delivery can be by bolusinjection, short term infusion or longer term infusion and can be viapassive delivery or through the utilisation of a suitable infusion pump.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats, co-solvents, organicsolvent mixtures, cyclodextrin complexation agents, emulsifying agents(for forming and stabilizing emulsion formulations), liposome componentsfor forming liposomes, gellable polymers for forming polymeric gels,lyophilisation protectants and combinations of agents for, inter alia,stabilising the active ingredient in a soluble form and rendering theformulation isotonic with the blood of the intended recipient.Pharmaceutical formulations for parenteral administration may also takethe form of aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents (R. G. Strickly,Solubilizing Excipients in oral and injectable formulations,Pharmaceutical Research, Vol 21(2) 2004, p 201-230).

A drug molecule that is ionizable can be solubilized to the desiredconcentration by pH adjustment if the drug's pK_(a) is sufficiently awayfrom the formulation pH value. The acceptable range is pH 2-12 forintravenous and intramuscular administration, but subcutaneously therange is pH 2.7-9.0. The solution pH is controlled by either the saltform of the drug, strong acids/bases such as hydrochloric acid or sodiumhydroxide, or by solutions of buffers which include but are not limitedto buffering solutions formed from glycine, citrate, acetate, maleate,succinate, histidine, phosphate, tris(hydroxymethyl)aminomethane (TRIS),or carbonate.

The combination of an aqueous solution and a water-soluble organicsolvent/surfactant (i.e., a cosolvent) is often used in injectableformulations. The water-soluble organic solvents and surfactants used ininjectable formulations include but are not limited to propylene glycol,ethanol, polyethylene glycol 300, polyethylene glycol 400, glycerin,dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP; Pharmasolve),dimethylsulphoxide (DMSO), Solutol HS 15, Cremophor EL, Cremophor RH 60,and polysorbate 80. Such formulations can usually be, but are notalways, diluted prior to injection.

Propylene glycol, PEG 300, ethanol, Cremophor EL, Cremophor RH 60, andpolysorbate 80 are the entirely organic water-miscible solvents andsurfactants used in commercially available injectable formulations andcan be used in combinations with each other. The resulting organicformulations are usually diluted at least 2-fold prior to IV bolus or IVinfusion.

Alternatively increased water solubility can be achieved throughmolecular complexation with cyclodextrins

Liposomes are closed spherical vesicles composed of outer lipid bilayermembranes and an inner aqueous core and with an overall diameter of <100μm. Depending on the level of hydrophobicity, moderately hydrophobicdrugs can be solubilized by liposomes if the drug becomes encapsulatedor intercalated within the liposome. Hydrophobic drugs can also besolubilized by liposomes if the drug molecule becomes an integral partof the lipid bilayer membrane, and in this case, the hydrophobic drug isdissolved in the lipid portion of the lipid bilayer. A typical liposomeformulation contains water with phospholipid at −5-20 mg/ml, anisotonicifier, a pH 5-8 buffer, and optionally cholesterol.

The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example water for injections, immediatelyprior to use.

The pharmaceutical formulation can be prepared by lyophilising acompound of formula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV),(IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereofas defined herein or acid addition salt thereof. Lyophilisation refersto the procedure of freeze-drying a composition. Freeze-drying andlyophilisation are therefore used herein as synonyms. A typical processis to solubilise the compound and the resulting formulation isclarified, sterile filtered and aseptically transferred to containersappropriate for lyophilisation (e.g. vials). In the case of vials, theyare partially stoppered with lyo-stoppers. The formulation can be cooledto freezing and subjected to lyophilisation under standard conditionsand then hermetically capped forming a stable, dry lyophile formulation.The composition will typically have a low residual water content, e.g.less than 5% e.g. less than 1% by weight based on weight of thelyophile.

The lyophilsation formulation may contain other excipients for example,thickening agents, dispersing agents, buffers, antioxidants,preservatives, and tonicity adjusters. Typical buffers includephosphate, acetate, citrate and glycine. Examples of antioxidantsinclude ascorbic acid, sodium bisulphite, sodium metabisulphite,monothioglycerol, thiourea, butylated hydroxytoluene, butylated hydroxylanisole, and ethylenediamietetraacetic acid salts. Preservatives mayinclude benzoic acid and its salts, sorbic acid and its salts, alkylesters of para-hydroxybenzoic acid, phenol, chlorobutanol, benzylalcohol, thimerosal, benzalkonium chloride and cetylpyridinium chloride.The buffers mentioned previously, as well as dextrose and sodiumchloride, can be used for tonicity adjustment if necessary.

Bulking agents are generally used in lyophilisation technology forfacilitating the process and/or providing bulk and/or mechanicalintegrity to the lyophilized cake. Bulking agent means a freely watersoluble, solid particulate diluent that when co-lyophilised with thecompound or salt thereof, provides a physically stable lyophilized cake,a more optimal freeze-drying process and rapid and completereconstitution. The bulking agent may also be utilised to make thesolution isotonic.

The water-soluble bulking agent can be any of the pharmaceuticallyacceptable inert solid materials typically used for lyophilisation. Suchbulking agents include, for example, sugars such as glucose, maltose,sucrose, and lactose; polyalcohols such as sorbitol or mannitol; aminoacids such as glycine; polymers such as polyvinylpyrrolidine; andpolysaccharides such as dextran.

The ratio of the weight of the bulking agent to the weight of activecompound is typically within the range from about 1 to about 5, forexample of about 1 to about 3, e.g. in the range of about 1 to 2.

Alternatively they can be provided in a solution form which may beconcentrated and sealed in a suitable vial. Sterilisation of dosageforms may be via filtration or by autoclaving of the vials and theircontents at appropriate stages of the formulation process. The suppliedformulation may require further dilution or preparation before deliveryfor example dilution into suitable sterile infusion packs.

Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets.

In one preferred embodiment of the invention, the pharmaceuticalcomposition is in a form suitable for i.v. administration, for exampleby injection or infusion.

Pharmaceutical compositions of the present invention for parenteralinjection can also comprise pharmaceutically acceptable sterile aqueousor nonaqueous solutions, dispersions, suspensions or emulsions as wellas sterile powders for reconstitution into sterile injectable solutionsor dispersions just prior to use.

Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), carboxymethylcellulose andsuitable mixtures thereof, vegetable oils (such as olive oil), andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the present invention may also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents, anddispersing agents. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents such as sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

If a compound is not stable in aqueous media or has low solubility inaqueous media, it can be formulated as a concentrate in organicsolvents. The concentrate can then be diluted to a lower concentrationin an aqueous system, and can be sufficiently stable for the shortperiod of time during dosing. Therefore in another aspect, there isprovided a pharmaceutical composition comprising a non aqueous solutioncomposed entirely of one or more organic solvents, which can be dosed asis or more commonly diluted with a suitable IV excipient (saline,dextrose; buffered or not buffered) before administration (Solubilizingexcipients in oral and injectable formulations, Pharmaceutical Research,21(2), 2004, p201-230). Examples of solvents and surfactants arepropylene glycol, PEG300, PEG400, ethanol, dimethylacetamide (DMA),N-methyl-2-pyrrolidone (NMP, Pharmasolve), Glycerin, Cremophor EL,Cremophor RH 60 and polysorbate. Particular non aqueous solutions arecomposed of 70-80% propylene glycol, and 20-30% ethanol. One particularnon aqueous solution is composed of 70% propylene glycol, and 30%ethanol. Another is 80% propylene glycol and 20% ethanol. Normally thesesolvents are used in combination and usually diluted at least 2-foldbefore IV bolus or IV infusion. The typical amounts for bolus IVformulations are ˜50% for Glycerin, propylene glycol, PEG300, PEG400,and ˜20% for ethanol. The typical amounts for IV infusion formulationsare ˜15% for Glycerin, 3% for DMA, and ˜10% for propylene glycol,PEG300, PEG400 and ethanol.

In one preferred embodiment of the invention, the pharmaceuticalcomposition is in a form suitable for i.v. administration, for exampleby injection or infusion. For intravenous administration, the solutioncan be dosed as is, or can be injected into an infusion bag (containinga pharmaceutically acceptable excipient, such as 0.9% saline or 5%dextrose), before administration.

In another preferred embodiment, the pharmaceutical composition is in aform suitable for sub-cutaneous (s.c.) administration.

Pharmaceutical dosage forms suitable for oral administration includetablets, capsules, caplets, pills, lozenges, syrups, solutions, powders,granules, elixirs and suspensions, sublingual tablets, wafers or patchesand buccal patches.

Pharmaceutical compositions containing compounds of the formula (I) canbe formulated in accordance with known techniques, see for example,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., USA.

Thus, tablet compositions can contain a unit dosage of active compoundtogether with an inert diluent or carrier such as a sugar or sugaralcohol, eg; lactose, sucrose, sorbitol or mannitol; and/or a non-sugarderived diluent such as sodium carbonate, calcium phosphate, calciumcarbonate, or a cellulose or derivative thereof such as methylcellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starchessuch as corn starch. Tablets may also contain such standard ingredientsas binding and granulating agents such as polyvinylpyrrolidone,disintegrants (e.g. swellable crosslinked polymers such as crosslinkedcarboxymethylcellulose), lubricating agents (e.g. stearates),preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents(for example phosphate or citrate buffers), and effervescent agents suchas citrate/bicarbonate mixtures. Such excipients are well known and donot need to be discussed in detail here.

Capsule formulations may be of the hard gelatin or soft gelatin varietyand can contain the active component in solid, semi-solid, or liquidform. Gelatin capsules can be formed from animal gelatin or synthetic orplant derived equivalents thereof.

The solid dosage forms (eg; tablets, capsules etc.) can be coated orun-coated, but typically have a coating, for example a protective filmcoating (e.g. a wax or varnish) or a release controlling coating. Thecoating (e.g. a Eudragit™ type polymer) can be designed to release theactive component at a desired location within the gastro-intestinaltract. Thus, the coating can be selected so as to degrade under certainpH conditions within the gastrointestinal tract, thereby selectivelyrelease the compound in the stomach or in the ileum or duodenum.

Instead of, or in addition to, a coating, the drug can be presented in asolid matrix comprising a release controlling agent, for example arelease delaying agent which may be adapted to selectively release thecompound under conditions of varying acidity or alkalinity in thegastrointestinal tract. Alternatively, the matrix material or releaseretarding coating can take the form of an erodible polymer (e.g. amaleic anhydride polymer) which is substantially continuously eroded asthe dosage form passes through the gastrointestinal tract. As a furtheralternative, the active compound can be formulated in a delivery systemthat provides osmotic control of the release of the compound. Osmoticrelease and other delayed release or sustained release formulations maybe prepared in accordance with methods well known to those skilled inthe art.

Compositions for topical use include ointments, creams, sprays, patches,gels, liquid drops and inserts (for example intraocular inserts). Suchcompositions can be formulated in accordance with known methods.

Compositions for parenteral administration are typically presented assterile aqueous or oily solutions or fine suspensions, or may beprovided in finely divided sterile powder form for making upextemporaneously with sterile water for injection.

Examples of formulations for rectal or intra-vaginal administrationinclude pessaries and suppositories which may be, for example, formedfrom a shaped moldable or waxy material containing the active compound.

Compositions for administration by inhalation may take the form ofinhalable powder compositions or liquid or powder sprays, and can beadministrated in standard form using powder inhaler devices or aerosoldispensing devices. Such devices are well known. For administration byinhalation, the powdered formulations typically comprise the activecompound together with an inert solid powdered diluent such as lactose.

The compounds of the formula (I) will generally be presented in unitdosage form and, as such, will typically contain sufficient compound toprovide a desired level of biological activity. For example, aformulation may contain from 1 nanogram to 2 grams of active ingredient,e.g. from 1 nanogram to 2 milligrams of active ingredient. Within thisrange, particular sub-ranges of compound are, or 0.1 milligrams to 2grams of active ingredient (more usually from 10 milligrams to 1 gram,e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams(for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2milligrams of active ingredient).

The active compound will be administered to a patient in need thereof(for example a human or animal patient) in an amount sufficient toachieve the desired therapeutic effect.

Where the compounds of the combination of the invention are presentedtogether, they can be formulated together as tablets, capsules,solutions for infusion or injection or any of the other solid or liquiddosage forms described above. For example, where they are formulatedtogether, they may be intimately mixed, or physically separated withinthe same formulation, for example by virtue of being present indifferent layers or granules within a tablet, or a separate beads orgranules within a capsule. More typically, however, they are formulatedseparately for separate or concurrent administration.

In one embodiment, the the individual components of the combination maybe formulated separately and presented together in the form of a kit,optionally under common outer packaging and optionally with instructionsfor their use.

More commonly these days, pharmaceutical formulations are prescribed toa patient in “patient packs” containing the whole course of treatment ina single package, usually a blister pack. Patient packs have anadvantage over traditional prescriptions, where a pharmacist divides apatient's supply of a pharmaceutical from a bulk supply, in that thepatient always has access to the package insert contained in the patientpack, normally missing in patient prescriptions. The inclusion of apackage insert has been shown to improve patient compliance with thephysicians instructions.

Accordingly, in a further embodiment, the invention provides a packagecontaining separate dosage units, one or more of which contain acompound of the formula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV),(IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereofas defined herein, and one or more of which contain a cytotoxic compoundor signalling inhibitor. Dosage units containing a compound of theformula (0), (I⁰), (1), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),(Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof as definedherein and a cytotoxic compound or signalling inhibitor have suitableamounts of active ingredient as defined herein. A package containsenough tablets, capsules or the like to treat a patient for apre-determined period of time, for instance for 2 weeks, 1 month or 3months.

Methods of Treatment

It is envisaged that the combinations containing a cytotoxic compound orsignalling inhibitor and compounds of the formula (0) and sub-groupsthereof such as formulae (I⁰), (I), (Ia), (Ib), (II), (III), (IV),(IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereofas defined herein will be useful in the prophylaxis or treatment of arange of disease states or conditions mediated by cyclin dependentkinases and/or GSKs (e.g. GSK-3). Examples of such disease states andconditions are set out herein.

The combinations are generally administered to a subject in need of suchadministration, for example a human or animal patient, preferably ahuman.

The compounds will typically be administered in amounts that aretherapeutically or prophylactically useful and which generally arenon-toxic. However, in certain situations (for example in the case oflife threatening diseases), the benefits of administering a compound ofthe formula (I) may outweigh the disadvantages of any toxic effects orside effects, in which case it may be considered desirable to administercompounds in amounts that are associated with a degree of toxicity.

The compounds may be administered over a prolonged term to maintainbeneficial therapeutic effects or may be administered for a short periodonly. Alternatively they may be administered in a pulsatile orcontinuous manner.

The compounds of the combination can be administered simultaneously orsequentially. When administered sequentially, they can be administeredat closely spaced intervals (for example over a period of 5-10 minutes)or at longer intervals (for example 1, 2, 3, 4 or more hours apart, oreven longer periods, e.g.1, 2, 3, 4, 5, 6,or 7 days, apart whererequired), the precise dosage regimen being commensurate with theproperties of the therapeutic agent(s). With sequential administration,the delay in administering the second (or additional) active ingredientshould not be such as to lose the advantageous benefit of theefficacious effect of the combination of the active ingredients. Inaddition, the delay in administering the second (or additional) activeingredient is typically timed so as to allow for any adverse sideeffects of the first compound to subside to an acceptable level beforeadminstration of the second compound, whilst not losing the advantageousbenefit of the efficacious effect of the combination of the activeingredients.

The two or more treatments may be given in individually varying doseschedules and via the same or different routes.

For example, one compound may be administered by the oral route and theother compound administered by parenteral administration such asadministration by injection (e.g. i.v.) or infusion. In an alternative,both compounds may be administered by injection or infusion. In afurther alternative, both compounds may be given orally. In oneparticular embodiment, the compound of the formula (I) is administeredby injection or infusion and the cytotoxic compound or signallinginhibitor is administered orally.

When administered at different times, the administration of onecomponent of the combination may alternate with or interleaf withadministration of the other component or the components of thecombination may be administered in sequential blocks of therapy. Asindicated above, the administration of the components of the combinationmay be spaced apart in time, for example by one or more hours, or days,or even weeks, provided that they form part of the same overalltreatment. In one embodiment of the invention, the compound of theformula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),(Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof as definedherein is administered sequentially or simultaneously with the cytotoxiccompound or signalling inhibitor.

In another embodiment of the invention, the compound of the formula (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa) (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof as defined herein isadministered sequentially with the cytotoxic compound or signallinginhibitor in either order.

In a further embodiment, the cytotoxic compound or signalling inhibitoris administered prior to the compound of the formula (0), (I⁰), (I),(Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein.

In a further embodiment, the taxane compound e.g. paclitaxel isadministered prior to the compound of the formula (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein.

In another embodiment, the cytotoxic compound or signalling inhibitor isadministered after the compound of the formula (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein.

In another embodiment of the invention, the compound of the formula (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof as defined herein and thecytotoxic compound or signalling inhibitor are administeredsimultaneously.

In another embodiment of the invention, the compound of the formula (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof as defined herein and thesignalling inhibitor are administered simultaneously.

In another embodiment of the invention, the compound of the formula (0),(I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa),(VIb), (VII) or (VIII) and sub-groups thereof as defined herein and thecytotoxic compound or signalling inhibitor are administeredsimultaneously.

In another embodiment, the compound of the formula (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein and the cytotoxiccompound or signalling inhibitor are each administered in atherapeutically effective amount with respect to the individualcomponents; in other words, the the compound of the formula (0), (I⁰),(I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb),(VII) or (VIII) and sub-groups thereof as defined herein and thecytotoxic compound or signalling inhibitor are administered in amountsthat would be therapeutically effective even if the components wereadministered other than in combination.

In another embodiment, the compound of the formula (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein and the cytotoxiccompound or signalling inhibitor are each administered in asub-therapeutic amount with respect to the individual components; inother words, the compound of the formula (0), (I⁰), (I), (Ia), (Ib),(II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof as defined herein and the cytotoxic compound orsignalling inhibitor are administered in amounts that would betherapeutically ineffective if the components were administered otherthan in combination.

Preferably, the cytotoxic compound or signalling inhibitor and thecompound of the formula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV),(IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereofas defined herein interact in a synergistic or additive manner.

Preferably, the cytotoxic compound or signalling inhibitor e.g.gemcitibine and the compound of the formula (0), (I⁰), (I), (Ia), (Ib),(II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof as defined herein interact in a synergistic oradditive manner.

Preferably, the taxane compound e.g. paclitaxel and the compound of theformula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),(Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof as definedherein interact in a synergistic or additive manner, and in particular asynergistic manner.

Preferably, the signalling inhibitor e.g. Iressa and the compound of theformula (0), (I⁰), (I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va),(Vb), (VIa), (VIb), (VII) or (VIII) and sub-groups thereof as definedherein interact in a synergistic or additive manner, and in particular asynergistic manner.

A typical daily dose of the compound of the formula (0), (I⁰), (I),(Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein can be in the range from100 picograms to 100 milligrams per kilogram of body weight, moretypically 5 nanograms to 25 milligrams per kilogram of bodyweight, andmore usually 10 nanograms to 15 milligrams per kilogram (e.g. 10nanograms to 10 milligrams, and more typically 1 microgram per kilogramto 20 milligrams per kilogram, for example 1 microgram to 10 milligramsper kilogram) per kilogram of bodyweight although higher or lower dosesmay be administered where required. The compound of the formula (I) canbe administered on a daily basis or on a repeat basis every 2, or 3, or4, or 5, or 6, or 7, or 10 or 14, or 21, or 28 days for example.

An example of a dosage for a 60 kilogram person comprises administeringa compound of the formula (I) as defined herein, for example the freebase of compound 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid piperidin-4-ylamide at a starting dosage of 4.5-10.8 mg/60kg/day(equivalent to 75-180 ug/kg/day) and subsequently by an efficacious doseof 44-97 mg/60 kg/day (equivalent to 0.7-1.6 mg/kg/day) or anefficacious dose of 72-274 mg/60 kg/day (equivalent to 1.2-4.6mg/kg/day). The mg/kg dose would scale pro-rata for any given bodyweight.

An example of a dosage for the mesylate salt is, at a starting dosage of5.6-13.5 mg/60 kg/day (equivalent to 93-225 μg/kg/day/person) andsubsequently by an efficacious dose of 55-122 mg/60 kg/day (equivalentto 0.9-2.0 mg/kg/day/person) or an efficacious dose of 90-345 mg/60kg/day (equivalent to 1.5-5.8 mg/kg/day/person).

In one particular dosing schedule, a patient will be given an infusionof a compound of the formula (I) for periods of one hour daily for up toten days in particular up to five days for one week, and the treatmentrepeated at a desired interval such as two to four weeks, in particularevery three weeks.

More particularly, a patient may be given an infusion of a compound ofthe formula (I) for periods of one hour daily for 5 days and thetreatment repeated every three weeks.

In another particular dosing schedule, a patient is given an infusionover 30 minutes to 1 hour followed by maintenance infusions of variableduration, for example 1 to 5 hours, e.g. 3 hours.

In a further particular dosing schedule, a patient is given a continuousinfusion for a period of 12 hours to 5 days, an in particular acontinuous infusion of 24 hours to 72 hours.

Ultimately, however, the quantity of compound administered, the type ofcomposition used, and the timing and frequency of the adminstration ofthe two components will be commensurate with the nature of the diseaseor physiological condition being treated and will be at the discretionof the physician.

Accordingly, a person skilled in the art would know through their commongeneral knowledge the dosing regimes and combination therapies to use.It will be appreciated that the preferred method and order ofadministration and the respective dosage amounts and regimes for eachcomponent of the combination will depend on the particular cytotoxiccompound or signalling inhibitor and compounds of formula (0), (I⁰),(I), (Ia), (Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb),(VII) or (VIII) and sub-groups thereof as defined herein beingadministered, their route of administration, the particular tumour beingtreated and the particular host being treated. The optimum method andorder of administration and dosage amounts and regime can be readilydetermined by those skilled in the art using conventional methods and inview of the information set out herein.

As described infra, the compounds of the formula (I) are administered incombination therapy with one of more other cytotoxic compounds, forexample in the treatment of a particular disease state (for example aneoplastic disease such as a cancer as hereinbefore defined). Examplesof suitable cytotoxic compounds that may be used in the combinations ofthe invention are described in detail above.

However, the combinations of the invention may also be further combinedwith other classes of therapeutic agents or treatments that may beadministered together (whether concurrently or at different timeintervals) with the combinations of the invention, including (but notlimited to):

-   -   1. hormones, hormone agonists, hormone antagonists and hormone        modulating agents (including antiandrogens, antiestrogens and        GNRAs);    -   2. monoclonal antibodies (e.g. monoclonal antibodies to cell        surface antigen(s));    -   3. alkylating agents (including aziridine, nitrogen mustard and        nitrosourea alkylating agents);    -   4. CDK inhibitors;    -   5. COX-2 inhibitors;    -   6. HDAC inhibitors;    -   7. DNA methylase inhibitors;    -   8. proteasome inhibitors;    -   9. Other therapeutic or prophylactic agents, for example agents        that reduce or alleviate some of the side effects associated        with chemotherapy. Particular examples of such agents include        anti-emetic agents and agents that prevent or decrease the        duration of chemotherapy-associated neutropenia and prevent        complications that arise from reduced levels of red blood cells        or white blood cells, for example erythropoietin (EPO),        granulocyte macrophage-colony stimulating factor (GM-CSF),        granulocyte-colony stimulating factor (G-CSF). In other        embodiments, the other therapeutic or prophylactic agents can be        as described below.

Alternatively, the combinations of the invention may also be furthercombined with other classes of therapeutic agents or treatments that maybe administered together (whether concurrently or at different timeintervals) with the combinations of the invention, including (but notlimited to):

-   -   1. hormones, hormone agonists, hormone antagonists and hormone        modulating agents (including antiandrogens, antiestrogens and        GNRAs);    -   2. monoclonal antibodies (e.g. monoclonal antibodies to cell        surface antigen(s));    -   3. camptothecin compounds;    -   4. antimetabolites;    -   5. vinca alkaloids;    -   6. taxanes;    -   7. platinum compounds;    -   8. DNA binders and Topo II inhibitors (including anthracycline        derivatives);    -   9. alkylating agents (including aziridine, nitrogen mustard and        nitrosourea alkylating agents);    -   10. a combination of two or more of the foregoing classes        (1)-(9).    -   11. signalling inhibitors (including PKB signalling pathway        inhibitors);    -   12. CDK inhibitors;    -   13. COX-2 inhibitors;    -   14. HDAC inhibitors;    -   15. DNA methylase inhibitors;    -   16. proteasome inhibitors;    -   17. a combination of two or more of the foregoing classes        (11)-(16);    -   18. a combination of two or more of the foregoing classes        (1)-(17);    -   19. Other therapeutic or prophylactic agents, for example agents        that reduce or alleviate some of the side effects associated        with chemotherapy. Particular examples of such agents include        anti-emetic agents and agents that prevent or decrease the        duration of chemotherapy-associated neutropenia and prevent        complications that arise from reduced levels of red blood cells        or white blood cells, for example erythropoietin (EPO),        granulocyte macrophage-colony stimulating factor (GM-CSF),        granulocyte-colony stimulating factor (G-CSF). In other        embodiments, the other therapeutic or prophylactic agents can be        as described below.

Other Therapeutic or Prophylactic Agents

The compositions may also include other therapeutic or prophylacticagents, for example agents that reduce or alleviate some of the sideeffects associated with chemotherapy. Particular examples of such agentsinclude anti-emetic agents and agents that prevent or decrease theduration of chemotherapy-associated neutropenia and preventcomplications that arise from reduced levels of red blood cells or whiteblood cells, for example erythropoietin (EPO), granulocytemacrophage-colony stimulating factor (GM-CSF), and granulocyte-colonystimulating factor (G-CSF).

Also included are agents that inhibit bone resorption such asbisphosphonate agents e.g. zoledronate, pamidronate and ibandronate, aswell as agents that suppress inflammatory responses (such asdexamethazone, prednisone, and prednisolone). Also included are agentsused to reduce blood levels of growth hormone and IGF-I in acromegalypatients such as synthetic forms of the brain hormone somatostatin,which includes octreotide acetate which is a long-acting octapeptidewith pharmacologic properties mimicking those of the natural hormonesomatostatin. Further included are agents such as leucovorin, which isused as an antidote to drugs that decrease levels of folic acid, orfolinic acid it self. In one particular embodiment is the combination of5FU and leucovorin or 5FU and folinic acid. In addition megestrolacetate can be used for the treatment of side-effects including oedemaand thromoembolic episodes.

Therefore in one embodiment the combinations further include anadditional agent selected from erythropoietin (EPO), granulocytemacrophage-colony stimulating factor (GM-CSF), granulocyte-colonystimulating factor (G-CSF), zoledronate, pamidronate, ibandronate,dexamethazone, prednisone, prednisolone, leucovorin, folinic acid andmegestrol acetate.

In particular the combinations further include an additional agentselected from erythropoietin (EPO), granulocyte macrophage-colonystimulating factor (GM-CSF), granulocyte-colony stimulating factor(G-CSF), zoledronate, pamidronate, dexamethazone, prednisone,prednisolone, leucovorin, and folinic acid such as erythropoietin (EPO),granulocyte macrophage-colony stimulating factor (GM-CSF) andgranulocyte-colony stimulating factor (G-CSF).

Zoledronic acid is available from Novartis under the Tradename Zometa®.It is used in the treatment of bone metastasis in a variety of tumortypes and for the treatment of hypercalcemia.

Pamidronate disodium (APD) available from Novartis under the tradenameAredia is a bone-resorption inhibitor and is used in the treatment ofmoderate or severe hypercalcemia. Pamidronate disodium is for i.v.injection.

Octreotide acetate is available from Novartis as Sandostatin LAR®(octreotide acetate for injectable suspension) and Sandostatin®(octreotide acetate for injection ampuls or for vials). Octreotide isknown chemically as L-Cysteinamide,D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxy-methyl)propyl]-, cyclic (2, 7)-disulfide; [R—(R*,R*)]. Synthetic forms of thebrain hormone somatostatin, such as octreotide, work at the site of thetumour. They bind to sst-2/sst-5 receptors to regulate gastrointestinalhormone secretion and affect tumour growth.

Each of the compounds present in the combinations of the invention maybe given in individually varying dose schedules and via differentroutes.

Thus, administration of the compound of the formula (I) in combinationtherapy with one or more cytotoxic compounds may comprise simultaneousor sequential administration. When administered sequentially, they canbe administered at closely spaced intervals (for example over a periodof 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or morehours apart, or even longer periods apart where required), the precisedosage regimen being commensurate with the properties of the therapeuticagent(s).

The combinations of the invention may also be administered inconjunction with non-chemotherapeutic treatments such as radiotherapy,photodynamic therapy, gene therapy, surgery and controlled diets.

The combination therapy may therefore involve the formulation of thecompound of the formula (I) with one, two, three, four or more othertherapeutic agents (including at least one cytotoxic compound orsignalling inhibitor). Such formulations can be, for example, a dosageform containing two, three, four or more therapeutic agents. In analternative, the individual therapeutic agents may be formulatedseparately and presented together in the form of a kit, optionally withinstructions for their use.

A person skilled in the art would know through their common generalknowledge the dosing regimes and combination therapies to use.

Methods of Diagnosis

Prior to administration of a compound of the formula (I), a patient maybe screened to determine whether a disease or condition from which thepatient is or may be suffering is one which would be susceptible totreatment with a compound having activity against cyclin dependentkinase(s) and/or GSK (e.g. GSK-3) or treatment with a cytotoxic compoundor signalling inhibitor.

For example, a biological sample taken from a patient may be analysed todetermine whether a condition or disease, such as cancer, that thepatient is or may be suffering from is one which is characterised by agenetic abnormality or abnormal protein expression which leads toover-activation of CDKs or to sensitisation of a pathway to normal CDKactivity. Examples of such abnormalities that result in activation orsensitisation of the CDK2 signal include up-regulation of cyclin E,(Harwell R M, Mull B B, Porter D C, Keyomarsi K.; J Biol Chem. 2004 Mar.26; 279(13):12695-705) or loss of p21 or p27, or presence of CDC4variants (Rajagopalan H, Jallepalli P V, Rago C, Velculescu V E, KinzlerK W, Vogelstein B, Lengauer C.; Nature. 2004 Mar. 4; 428(6978):77-81).The term up-regulation includes elevated expression or over-expression,including gene amplification (i.e. multiple gene copies) and increasedexpression by a transcriptional effect, and hyperactivity andactivation, including activation by mutations. Thus, the patient may besubjected to a diagnostic test to detect a marker characteristic ofup-regulation of cyclin E, or loss of p21 or p27, or presence of CDC4variants. The term diagnosis includes screening. By marker we includegenetic markers including, for example, the measurement of DNAcomposition to identify mutations of CDC4. The term marker also includesmarkers which are characteristic of up regulation of cyclin E, includingenzyme activity, enzyme levels, enzyme state (e.g. phosphorylated ornot) and mRNA levels of the aforementioned proteins.

Tumours with upregulation of cyclin E, or loss of p21 or p27 may beparticularly sensitive to CDK inhibitors. Tumours may preferentially bescreened for upregulation of cyclin E, or loss of p21 or p27 prior totreatment. Thus, the patient may be subjected to a diagnostic test todetect a marker characteristic of up-regulation of cyclin E, or loss ofp21 or p27. The diagnostic tests are typically conducted on a biologicalsample selected from tumour biopsy samples, blood samples (isolation andenrichment of shed tumour cells), stool biopsies, sputum, chromosomeanalysis, pleural fluid, peritoneal fluid, or urine.

It has been found, Rajagopalan et al (Nature. 2004 Mar. 4;428(6978):77-81), that there were mutations present in CDC4 (also knownas Fbw7 or Archipelago) in human colorectal cancers and endometrialcancers (Spruck et al, Cancer Res. 2002 Aug. 15; 62(16):4535-9).Identification of individual carrying a mutation in CDC4 may mean thatthe patient would be particularly suitable for treatment with a CDKinhibitor. Tumours may preferentially be screened for presence of a CDC4variant prior to treatment. The screening process will typically involvedirect sequencing, oligonucleotide microarray analysis, or a mutantspecific antibody.

Methods of identification and analysis of mutations and up-regulation ofproteins are known to a person skilled in the art. Screening methodscould include, but are not limited to, standard methods such asreverse-transcriptase polymerase chain reaction (RT-PCR) or in-situhybridisation.

In screening by RT-PCR, the level of mRNA in the tumour is assessed bycreating a cDNA copy of the mRNA followed by amplification of the cDNAby PCR. Methods of PCR amplification, the selection of primers, andconditions for amplification, are known to a person skilled in the art.Nucleic acid manipulations and PCR are carried out by standard methods,as described for example in Ausubel, F. M. et al., eds. CurrentProtocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis,M. A. et-al., eds. PCR Protocols: a guide to methods and applications,1990, Academic Press, San Diego. Reactions and manipulations involvingnucleic acid techniques are also described in Sambrook et al., 2001,3^(rd) Ed, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press. Alternatively a commercially available kit for RT-PCR(for example Roche Molecular Biochemicals) may be used, or methodologyas set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;5,192,659, 5,272,057, 5,882,864, and 6,218,529 and incorporated hereinby reference.

An example of an in-situ hybridisation technique for assessing mRNAexpression would be fluorescence in-situ hybridisation (FISH) (seeAngerer, 1987 Meth. Enzymol., 152: 649).

Generally, in situ hybridization comprises the following major steps:(1) fixation of tissue to be analyzed; (2) prehybridization treatment ofthe sample to increase accessibility of target nucleic acid, and toreduce nonspecific binding; (3) hybridization of the mixture of nucleicacids to the nucleic acid in the biological structure or tissue; (4)post-hybridization washes to remove nucleic acid fragments not bound inthe hybridization, and (5) detection of the hybridized nucleic acidfragments. The probes used in such applications are typically labeled,for example, with radioisotopes or fluorescent reporters. Preferredprobes are sufficiently long, for example, from about 50, 100, or 200nucleotides to about 1000 or more nucleotides, to enable specifichybridization with the target nucleic acid(s) under stringentconditions. Standard methods for carrying out FISH are described inAusubel, F. M. et al., eds. Current Protocols in Molecular Biology,2004, John Wiley & Sons Inc and Fluorescence In Situ Hybridization:Technical Overview by John M. S. Bartlett in Molecular Diagnosis ofCancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004,pps. 077-088; Series: Methods in Molecular Medicine.

Alternatively, the protein products expressed from the mRNAs may beassayed by immunohistochemistry of tumour samples, solid phaseimmunoassay with microtiter plates, Western blotting, 2-dimensionalSDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and othermethods known in the art for detection of specific proteins. Detectionmethods would include the use of site specific antibodies. The skilledperson will recognize that all such well-known techniques for detectionof upregulation of cyclin E, or loss of p21 or p27, or detection of CDC4variants could be applicable in the present case.

Therefore all of these techniques could also be used to identify tumoursparticularly suitable for treatment with combinations of CDK inhibitorsand cytotoxic compounds or signalling inhibitors. Patients with mantlecell lymphoma (MCL) could be selected for treatment with a CDK inhibitorusing diagnostic tests outlined herein. MCL is a distinctclinicopathologic entity of non-Hodgkin's lymphoma, characterized byproliferation of small to medium-sized lymphocytes with co-expression ofCD5 and CD20, an aggressive and incurable clinical course, and frequentt(11;14)(q13;q32) translocation. Over-expression of cyclin D1 mRNA,found in mantle cell lymphoma (MCL), is a critical diagnostic marker.Yatabe et al (Blood. 2000 Apr. 1; 95(7):2253-61) proposed that cyclinD1-positivity should be included as one of the standard criteria forMCL, and that innovative therapies for this incurable disease should beexplored on the basis of the new criteria. Jones et al (J Mol Diagn.2004 May; 6(2):84-9) developed a real-time, quantitative, reversetranscription PCR assay for cyclin D1 (CCND1) expression to aid in thediagnosis of mantle cell lymphoma (MCL). Howe et al (Clin Chem. 2004January; 50(1):80-7) used real-time quantitative RT-PCR to evaluatecyclin D1 mRNA expression and found that quantitative RT-PCR for cyclinD1 mRNA normalized to CD19 mRNA can be used in the diagnosis of MCL inblood, marrow, and tissue. Alternatively, patients with breast cancercould be selected for treatment with a CDK inhibitor using diagnostictests outline above. Tumour cells commonly overexpress cyclin E and ithas been shown that cyclin E is over-expressed in breast cancer (Harwellet al, Cancer Res, 2000, 60, 481-489). Therefore breast cancer may inparticular be treated with a CDK inhibitor.

Examples

The invention will now be illustrated, but not limited, by reference tothe specific embodiments described in the following examples.

In the examples, the compounds prepared were characterised by liquidchromatography and mass spectroscopy (LC-MS) using the system andoperating conditions set out below. Where chlorine is present and asingle mass is quoted, the mass quoted for the compound is for ³⁵Cl. Thetwo systems were equipped with identical chromatography columns and wereset up to run under the same operating conditions. The operatingconditions used are also described below. In the examples, the retentiontimes are given in minutes.

Platform System

System: Waters 2790/Platform LC

Mass Spec Detector: Micromass Platform LC

PDA Detector: Waters 996 PDA

Analytical Conditions:

Eluent A: 5% CH3CN in 95% H₂O (0.1% Formic Acid)

Eluent B: CH₃CN (0.1% Formic Acid)

Gradient: 10-95% eluent B

Flow: 1.2 ml/min

Column: Synergi 4 μm Max-RP C₁₂, 80A, 50×4.6 mm (Phenomenex)

MS Conditions:

Capillary voltage: 3.5 kV

Cone voltage: 30 V

Source Temperature: 120° C.

FractionLynx System

System: Waters FractionLynx (dual analytical/prep)

Mass Spec Detector: Waters-Micromass ZQ

PDA Detector: Waters 2996 PDA

Analytical Conditions:

Eluent A: H₂O (0.1% Formic Acid)

Eluent B: CH₃CN (0.1% Formic Acid)

Gradient: 5-95% eluent B

Flow: 1.5 ml/min

Column: Synergi 4 μm Max-RP C₁₂, 80A, 50×4.6 mm (Phenomenex)

MS Conditions:

Capillary voltage: 3.5 kV

Cone voltage: 30 V

Source Temperature: 120° C.

Desolvation Temperature: 300° C.

Analytical LC-MS System

Several systems were used, as described below, and these were equippedwith were set up to run under closely similar operating conditions. Theoperating conditions used are also described below.

HPLC System: Waters 2795

Mass Spec Detector: Micromass Platform LC

PDA Detector: Waters 2996 PDA

Acidic Analytical Conditions:

Eluent A: H₂O (0.1% Formic Acid)

Eluent B: CH₃CN (0.1% Formic Acid)

Gradient: 5-95% eluent B over 3.5 minutes

Flow: 0.8 ml/min

Column: Phenomenex Synergi 4 μ MAX-RP 80A, 2.0×50 mm

Basic Analytical Conditions:

Eluent A: H₂O (10 mM NH₄HCO₃ buffer adjusted to pH=9.5 with NH₄OH)

Eluent B: CH₃CN

Gradient: 05-95% eluent B over 3.5 minutes

Flow: 0.8 ml/min

Column: Thermo Hypersil-Keystone BetaBasic-18 5 μm 2.1×50 mm

or

Column: Phenomenex Luna C18(2) 5 μm 2.0×50 mm

Polar Analytical Conditions: Eluent A: H₂O (0.1% Formic Acid)

Eluent B: CH₃CN (0.1% Formic Acid)

Gradient: 00-50% eluent B over 3 minutes

Flow: 0.8 ml/min

Column: Thermo Hypersil-Keystone HyPurity Aquastar, 5μ, 2.1×50 mm

or

Column: Phenomenex Synergi 4μ MAX-RP 80A, 2.0×50 mm or

Longer Analytical Conditions:

Eluent A: H₂O (0.1% Formic Acid)

Eluent B: CH₃CN (0.1% Formic Acid)

Gradient: 05-95% eluent B over 15 minutes

Flow: 0.4 ml/min

Column: Phenomenex Synergi 4μ MAX-RP 80A, 2.0×150 mm

MS Conditions:

Capillary voltage: 3.6 kV

Cone voltage: 30 V

Source Temperature: 120° C.

Scan Range: 165-700 amu

Ionisation Mode: ElectroSpray Positive or

-   -   ElectroSpray Negative or    -   ElectroSpray Positive & Negative

Mass Directed Purification LC-MS System

The following preparative chromatography systems can be used to purifythe compounds of the invention.

Hardware:

Waters Fractionlynx System:

2767 Dual Autosampler/Fraction Collector

2525 preparative pump

CFO (column fluidic organiser) for column selection

RMA (Waters reagent manager) as make up pump

Waters ZQ Mass Spectrometer

Waters 2996 Photo Diode Array detector

Software: Masslynx 4.0

Columns:

1. Low pH chromatography: Phenomenex Synergy MAX-RP, 10μ, 150×15mm(alternatively used same column type with 100×21.2mm dimensions).

2. High pH chromatography: Phenomenex Luna C18 (2), 10μ, 100×21.2 mm(alternatively used Thermo Hypersil Keystone BetaBasic C18, 5μ, 100×21.2mm)

Eluents:

1. Low pH chromatography:

Solvent A: H₂0+0.1% Formic Acid, pH 1.5

Solvent B: CH₃CN+0.1% Formic Acid

2. High pH chromatography:

Solvent A: H₂0+10 mM NH₄HCO₃+NH₄OH, pH 9.5

Solvent B: CH3CN

3. Make up solvent: MeOH +0.1% formic acid (for both chromatographytype)

Methods:

Prior to using preparative chromatography to isolate and purify theproduct compounds, analytical LC-MS (see above) can first be used todetermine the most appropriate conditions for preparativechromatography. A typical routine is to run an analytical LC-MS usingthe type of chromatography (low or high pH) most suited for compoundstructure. Once the analytical trace shows good chromatography, asuitable preparative method of the same type can be chosen. Typicalrunning condition for both low and high pH chromatography methods are:

Flow rate: 24 ml/min

Gradient: Generally all gradients have an initial 0.4 min step with 95%A+5% B. Then according to analytical trace a 3.6 min gradient is chosenin order to achieve good separation (e.g. from 5% to 50% B for earlyretaining compounds; from 35% to 80% B for middle retaining compoundsand so on)

Wash: 1 minute wash step is performed at the end of the gradient

Re-equilibration: A 2.1 minute re-equilibration step is carried out toprepare the system for the next run

Make Up flow rate: 1 ml/min

Solvent:

All compounds were usually dissolved in 100% MeOH or 100% DMSO

MS Running Conditions:

Capillary voltage: 3.2 kV

Cone voltage: 25 V

Source Temperature: 120° C.

Multiplier: 500 V

Scan Range: 125-800 amu

Ionisation Mode: ElectroSpray Positive

The starting materials for each of the Examples are commerciallyavailable unless otherwise specified.

Example 1 4-Amino-1 H-pyrazole-3-carboxylic acid phenylamide 1A.4-Nitro-1H-pyrazole-3-carboxylic acid phenylamide

4-Nitropyrazole-3-carboxylic acid (2.5 g; 15.9 mmol) was added to astirred solution of aniline (1.6 ml; 17.5 mmol), EDC (3.7 g; 19.1 mmol),and HOBt (2.6 g; 19.1 mmol) in N,N-dimethylformamide (DMF) (25 ml), thenstirred at room temperature overnight. The solvent was removed byevaporation under reduced pressure and the residue triturated with ethylacetate/saturated NaHCO₃ solution. The resultant solid was collected byfiltration, washed with water and diethyl ether then dried under vacuumto give 2.85 g of the title compound (sodium salt) as a yellow/brownsolid. (LC/MS: R_(t) 2.78, [M+H]⁺ 232.95).

1B. 4-Amino-1 H-pyrazole-3-carboxylic acid phenylamide

4-Nitro-1H-pyrazole-3-carboxylic acid phenylamide (100 mg; 0.43 mmol)was dissolved in ethanol (5 ml), treated with tin (II) chloridedihydrate (500 mg; 2.15 mmol) then heated at reflux overnight. Thereaction mixture was cooled and evaporated. The residue was partitionedbetween ethyl acetate and brine, and the ethyl acetate layer wasseparated, dried (MgSO₄), filtered and evaporated. The crude product waspurified by flash column chromatography eluting with 1:1 ethylacetate/petroleum ether then 5% methanol/dichloromethane. Evaporation ofproduct containing fractions followed by preparative LC/MS gave 15 mg ofthe product as an off white solid. (LC/MS: R_(t) 1.40, [M+H]⁺ 202.95).

Example 2 4-Acetylamino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide 2A. 4-Nitro-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

4-Nitropyrazole-3-carboxylic acid (10 g; 63.66 mmol) was added to astirred solution of 4-fluoroaniline (6.7 ml; 70 mmol), EDC (14.6 g; 76.4mmol), and HOBt (10.3 g; 76.4 mmol) in DMF (25 ml), then stirred at roomtemperature overnight. The solvent was removed by evaporation underreduced pressure and the residue triturated with ethyl acetate/saturatedbrine solution. The resultant yellow solid was collected by filtration,washed with 2M hydrochloric acid, then dried under vacuum to give 15.5 gof the title compound. (LC/MS: R_(t) 2.92 [M+H]⁺ 250.89).

2B. 4-Amino-1H-pyrazole-3-carboxylic acid (4-fluoro-phenyl)-amide

4-Nitro-1H-pyrazole-3-carboxylic acid (4-fluorophenyl)-amide (15 g) wasdissolved in 200 ml of ethanol, treated with 1.5 g of 10% palladium oncarbon under a nitrogen atmosphere, then hydrogenated at roomtemperature and pressure overnight. The catalyst was removed byfiltration through Celite and the filtrate evaporated. The crude productwas dissolved in acetone/water (100 m1:100 ml) and after slowevaporation of the acetone the product was collected by filtration as abrown crystalline solid (8.1 g). (LC/MS: R_(t) 1.58, [M+H]⁺ 220.95).

2C. 4-Acetylamino-1H-pyrazole-3-carboxylic acid (4-fluoro-phenyl)-amide

4-Amino-1H-pyrazole-3-carboxylic acid (4-fluorophenyl)-amide (500 mg;2.27 mmol) was dissolved in 5 ml of pyridine, treated with aceticanhydride (240 μl, 2.5 mmol) then stirred at room temperature overnight.The solvent was removed by evaporation then dichloromethane (20 ml) and2M hydrochloric acid (20 ml) were added. The undissolved solid wascollected by filtration, washed with more dichloromethane and water thendried under vacuum. The product was isolated as an off white solid (275mg). (LC/MS: R_(t) 2.96, [M+H]⁺ 262.91).

Example 3 4-(2,2,2-Trifluoro-acetylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

4-Amino-1H-pyrazole-3-carboxylic acid (4-fluorophenyl)-amide (Example2B) (500 mg; 2.27 mmol) was dissolved in 5 ml of pyridine, treated withtrifluoroacetic anhydride (320 μl, 2.5 mmol) then stirred at roomtemperature overnight. The solvent was removed by evaporation, theresidue was partitioned between ethyl acetate (50 ml) and 2 Mhydrochloric acid (50 ml), and the ethyl acetate layer was separated,washed with brine (50 ml), dried (MgSO₄), filtered and evaporated togive 560 mg of product as a brown solid. (LC/MS: [M+H]⁺ 317).

Example 44-[(5-Oxo-pyrrolidine-2-carbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

To a stirred solution of 4-amino-1H-pyrazole-3-carboxylic acid(4-fluorophenyl)-amide (Example 2B) (50 mg; 0.23 mmol), EDAC (52 mg;0.27 mmol) and HOBt (37 mg; 0.27 mmol) in 5 ml of DMF was added2-oxoproline (33 mg; 0.25 mmol), and the mixture was then left at roomtemperature overnight. The reaction mixture was evaporated and theresidue purified by preparative LC/MS, to give 24 mg of the product as awhite solid. (LC/MS: R_(t) 2.27 [M+H]⁺ 332).

Example 5 4-Phenylacetylamino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4 butusing phenylacetic acid (34 mg; 0.23 mmol) as the starting material. Thetitle compound (14 mg) was isolated as a white solid. (LC/MS: R_(t) 3.24[M+H]⁺ 339).

Example 6 4-(2-1H-Indol-3-yl-acetylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing indole-3-acetic acid (44 mg; 0.23 mmol) as the starting material.The title product (14 mg) was isolated as a white solid. (LC/MS: R_(t)3.05 [M+H]⁺ 378).

Example 7 4-(2-Benzenesulphonyl-acetylamino)-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 2-(phenylsulphonyl) acetic acid (50 mg; 0.23 mmol) as the startingmaterial. The title compound (29 mg) was isolated as a white solid.(LC/MS: R_(t) 3.00 [M+H]⁺ 403).

Example 84-[2-(5-Amino-tetrazol-1-yl)-acetylamino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, but5-aminotetrazole-1-acetic acid (36 mg; 0.23 mmol) was used as thestarting material. The title compound (23 mg) was isolated as a whitesolid. (LC/MS: R_(t) 2.37 [M+H]⁺ 346).

Example 9N-[3-(4-Fluoro-phenylcarbamoyl)-1H-pyrazol-4-yl]-6-hydroxy-nicotinamide

The reaction was carried out in a manner analogous to Example 4, butusing 6-hydroxynicotinic acid (38 mg; 0.23 mmol) as the startingmaterial. The title compound (17 mg) was isolated as a white solid.(LC/MS: R_(t) 2.32 [M+H]⁺ 342).

Example 104-[3-(4-Chloro-phenyl)-propionylamino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 3-(4-chlorophenyl)propionic acid (46 mg; 0.23 mmol) as thestarting material. The title compound (40 mg) was isolated as a whitesolid. (LC/MS: R_(t) 3.60 [M+H]⁺ 388).

Example 114-(3-4H-[1,2,4]Triazol-3-yl-propionylamino)-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 3-triazol-3-yl propionic acid (36 mg; 0.23 mmol) as the startingmaterial. The title compound (18 mg) was isolated as a white solid.(LC/MS: R_(t) 2.39 [M+H]⁺ 344).

Example 124-[2-(1-Methyl-1H-indol-3-yl)-acetylamino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing N-methyl indole-3-acetic acid (48 mg; 0.23 mmol) as the startingmaterial. The title compound (20 mg) was isolated as a white solid.(LC/MS: R_(t) 3.34 [M+H]⁺ 392).

Example 134-[(1-Hydroxy-cyclopropanecarbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 1-hydroxycyclopropane carboxylic acid (26 mg; 0.23 mmol) as thestarting material. The title compound (24 mg) was isolated as a whitesolid. (LC/MS: R_(t) 2.55 [M+H]⁺ 305).

Example 14 1-Acetyl-piperidine-4-carboxylic acid[3-(4-fluoro-phenylcarbamoyl)-1H-pyrazol-4-yl]-amide

The reaction was carried out in a manner analogous to Example 4, butusing N-acetylpiperidine acetic acid (43 mg; 0.23 mmol) as the startingmaterial. The title compound (19 mg) was isolated as a white solid.(LC/MS: R_(t) 2.49 [M+H]⁺ 374).

Example 154-[3-(4-Methyl-piperazin-1-yl)-propionylamino]-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 4-N-methylpiperazine-1-N-propionic acid (31 mg; 0.23 mmol) as thestarting material. The title compound (19 mg) was isolated as a whitesolid. (LC/MS: R_(t) 1.77 [M+H]⁺ 375).

Example 16 4-(2-1H-Imidazol-4-yl-acetylamino)-1H-pyrazole-3-carboxylicacid (4-fluorophenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing imidazole-4-acetic acid (32 mg; 0.23 mmol) as the startingmaterial. The title compound (35 mg) was isolated as a white solid.(LC/MS: R_(t) 1.82 [M+H]⁺ 329).

Example 17 4-(3-Morpholin-4-yl-propionylamino)-1H-pyrazole-3-carboxylicacid (4-fluorophenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 3-morpholin-4-yl-propionic acid (40 mg; 0.23 mmol) as the startingmaterial. The title compound (15 mg) was isolated as a white solid.(LC/MS: R_(t) 1.84 [M+H]⁺ 362).

Example 18 4-(3-Piperidin-1-yl-propionylamino)-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide

The reaction was carried out in a manner analogous to Example 4, butusing 3-piperidine-4-yl-propionic acid (39 mg; 0.23 mmol) as thestarting material. The title compound (19 mg) was isolated as a whitesolid. (LC/MS: R_(t) 1.92 [M+H]⁺ 360).

Example 19 4-Cyclohexylamino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

To a solution of 4-amino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide (200 mg; 1 mmol) and cyclohexanone (107 mg; 1.1mmol) in dichloromethane (10 ml) were added 3A molecular sieves (1 g)and sodium triacetoxyborohydride (315 mg; 1.5 mmol), and the mixture wasthen stirred at room temperature over the weekend. The reaction mixturewas filtered through Celite®, diluted with ethyl acetate, washed withbrine, dried (MgSO₄) and evaporated to give the 48 mg of the product asa grey gum. (LC/MS: R_(t) 2.95, [M+H]⁺ 285).

Example 20 4-Isopropylamino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The title compound was prepared in a manner analogous to Example 19, butusing acetone in place of cyclohexanone. (LC/MS: R_(t) 2.08, [M+H]⁺245).

Example 21 4-(2-Hydroxy-1-methyl-ethylamino)-1H-pyrazole-3-carboxylicacid (4-fluorophenyl)-amide

The compound was prepared in a manner analogous to Example 19, but usinghydroxyacetone in place of cyclohexanone. ¹HNMR (400 MHz, D6-DMSO): 9.9(1H, br s), 7.8 (2H, dd), 7.3 (1H, s), 7.15 (2H, t), 5.15 (1H, d), 4.7(1H, br s), 3.4 (2H, m), 3.2 (1H, m), 1.1 (3H, d).

Example 22 4-(1-Ethyl-propylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The compound was prepared in a manner analogous to Example 19, but using3-pentanone in place of cyclohexanone. ¹HNMR (400 MHz, D6-DMSO): 12.85(1h,br s), 9.9 (1H, br s), 7.8 (2H, br t), 7.3 (1H, s), 7.15 (2H, t),5.0 (1H, d), 2.9 (1H, br m), 1.5 (4H, m), 3.2 (1H, m), 0.9 (6H, t).

Example 23 4-(3-Chloro-pyrazin-2-ylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

A mixture of 4-amino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide (50 mg; 0.23 mmol) and 2,3-dichloropyrazine (140mg; 0.92 mmol) was heated at 150° C. (50W) for 20 minutes in a CEMDiscover™ microwave synthesiser. The crude reaction mixture was purifiedby flash column chromatography eluting with ethyl acetate/hexane (1:3then 1:2). Product containing fractions were combined and evaporated togive 15 mg of the title compound as a white solid. (LC/MS: R_(t) 4.06M+H]⁺ 332).

Example 24 4-(Pyrazin-2-ylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The compound was prepared in a manner analogous to Example 23, but using2-chloropyrazine in place of 2,3-dichloropyrazine. (LC/MS: R_(t) 3.28[M+H]⁺ 299).

Example 25 Synthesis of4-(2-Methoxy-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

2-Methoxy-benzoic acid (38 mg, 0.25 mmol) was added to a solution of4-amino-1H-pyrazole-3-carboxylic acid (4-fluoro-phenyl)-amide (50 mg,0.23 mmol), EDC (53 mg, 0.27 mmol), and HOBt (37 mg, 0.27 mmol) in DMF(5 ml). The reaction mixture was stirred at room temperature for 24hours. The solvent was removed under reduced pressure. The residue waspurified by preparative LC/MS and, after evaporation ofproduct-containing fractions, yielded the product as a pinkish solid (12mg, 15%). (LC/MS: R_(t) 4.00, [M+H]⁺ 354.67).

Example 26 Synthesis of 4-Benzoylamino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example25 using benzoic acid (31 mg, 0.25 mmol) as starting acid. The productwas isolated as a pink solid (26 mg, 35%). (LC/MS: R_(t) 3.96, [M+H]⁺324.65).

Example 27 Synthesis of4-(Cyclohexanecarbonyl-amino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example25 using cyclohexanecarboxylic acid (32 mg, 0.25 mmol) as starting acid.The product was isolated as a pink solid (28 mg, 37%). (LC/MS: R_(t)4.16, [M+H]⁺ 330.70).

Example 28 Synthesis of4-[(1-Methyl-cyclopropanecarbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example25 using 1-methyl-cyclopropanecarboxylic acid (25 mg, 0.25 mmol) asstarting acid. The product was isolated as a pink solid (24 mg, 35%).(LC/MS: R_(t) 3.72, [M+H]⁺ 302.68).

Example 29 Synthesis of4-(2-Hydroxy-acetylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example25 using hydroxy-acetic acid (19 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a white solid (26 mg, 41%). (LC/MS: R_(t) 2.65,[M+H]⁺ 278.61).

Example 30 Synthesis of4-(2,2-Dimethyl-propionylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example25 using 2,2-dimethyl-propionic acid (26 mg, 0.25 mmol) as startingacid. The product was isolated as a pink solid (21 mg, 30%). (LC/MS:R_(t) 3.83, [M+H]⁺ 304.68).

Example 31 Synthesis of4-(3-Hydroxy-propionylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example25 using 3-hydroxy-propionic acid (75.1 mg, 0.25 mmol) as starting acid.The product was isolated as a beige solid (5 mg, 8%). (LC/MS: R_(t)2.58, [M+H]⁺ 292.65).

Example 32 Synthesis of4-(2-Fluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

2-Fluorobenzoic acid (36 mg, 0.25 mmol) was added to a solution of4-amino-1H-pyrazole-3-carboxylic acid (4-fluoro-phenyl)-amide (50 mg,0.23 mmol), EDC (53 mg, 0.27 mmol) and HOBt (37 mg, 0.27 mmol) in DMSO(1 ml). The reaction mixture was stirred at room temperature for 24hours and purified by preparative LC/MS. Evaporation ofproduct-containing fractions yielded the product as a white solid (15mg, 19%). (LC/MS: R_(t) 3.91, [M+H]⁺ 342.66).

Example 33 Synthesis of4-(3-Fluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 3-fluorobenzoic acid (36 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a white solid (19 mg, 24%). (LC/MS: R_(t) 4.03,[M+H]⁺ 342.67).

Example 34 Synthesis of4-(3-Methoxy-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 3-methoxy-benzoic acid (39 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a white solid (20 mg, 25%). (LC/MS: R_(t) 3.97,[M+H]⁺ 354.68).

Example 35 Synthesis of4-(2-Nitro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 2-nitrobenzoic acid (43 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a white solid (17 mg, 20%). (LC/MS: R_(t) 3.67,[M+H]⁺ 369.66).

Example 36 Synthesis of4-(4-Nitro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 4-nitrobenzoic acid (43 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a white solid (15 mg, 18%). (LC/MS: R_(t) 3.98,[M+H]⁺ 369.63).

Example 37 Synthesis of4-[(3-Methyl-furan-2-carbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 3-methyl-2-furoic acid (32 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a white solid (15 mg, 20%). (LC/MS: R_(t) 3.86,[M+H]⁺ 328.68).

Example 38 Synthesis of4-[(Furan-2-carbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 2-furoic acid (29 mg, 0.25 mmol) as starting acid. The productwas isolated as a white solid (18 mg, 25%). (LC/MS: R_(t) 3.56, [M+H]⁺314.64).

Example 39 Synthesis of4-[(3H-Imidazole-4-carbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 1H-imidazole-4-carboxylic acid (29 mg, 0.25 mmol) as startingacid. The product was isolated as a white solid (16 mg, 22%). (LC/MS:R_(t) 2.59, [M+H]⁺ 314.65).

Example 40 Synthesis of4-(4-Fluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 4-fluorobenzoic acid (36 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a cream coloured solid (23 mg, 29%). (LC/MS:R_(t) 4.00, [M+H]⁺ 342.67).

Example 41 Synthesis of4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 2,6-difluorobenzoic acid (40 mg, 0.25 mmol) as starting acid.The product was isolated as a cream coloured solid (25 mg, 30%). (LC/MS:R_(t) 3.76, [M+H]⁺ 360.66).

Example 42 Synthesis of4-(3-Nitro-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 3-nitrobenzoic acid (43 mg, 0.25 mmol) as starting acid. Theproduct was isolated as a cream coloured solid (15 mg, 18%). (LC/MS:R_(t) 3.94, [M+H]⁺ 369.65).

Example 43 Synthesis of 1H-Indole-3-carboxylic acid[3-(4-fluoro-phenylcarbamoyl)-1H-pyrazol-4-yl]-amide

The experiment was carried out in a manner analogous to that of Example32 using indole-3-carboxylic acid (41 mg, 0.25 mmol) as starting acid.The product was isolated as a rust coloured solid (14 mg, 17%). (LC/MS:R_(t) 3.60, [M+H]⁺ 363.66).

Example 44 Synthesis of4-(4-Hydroxymethyl-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 4-hydroxymethylbenzoic acid (39 mg, 0.25 mmol) as startingacid. The product was isolated as a white solid (19 mg, 23%). (LC/MS:R_(t) 3.12, [M+H]⁺ 354.68).

Example 45 Synthesis of4-(3-Methyl-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 3-methylbenzoic acid (35 mg, 0.25 mmol) as starting acid. Theproduct was isolated as an off-white solid (21 mg, 27%). (LC/MS: R_(t)4.13, [M+H]⁺ 338.71).

Example 46 Synthesis of4-(2-Methyl-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 2-methylbenzoic acid (35 mg, 0.25 mmol) as starting acid. Theproduct was isolated as an off-white solid (20 mg, 26%). (LC/MS: R_(t)4.05, [M+H]⁺ 338.69).

Example 47 Synthesis of4-(4-Methyl-benzoylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example32 using 4-methylbenzoic acid (35 mg, 0.25 mmol) as starting acid. Theproduct was isolated as an off-white solid (19 mg, 24%). (LC/MS: R_(t)4.16, [M+H]⁺ 338.70).

Example 48 Synthesis of4-[(2-Methyl-thiophene-3-carbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

2-Methyl-3-thiophenecarboxylic acid (36 mg, 0.25 mmol) was added to asolution of 4-amino-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide (Example 2B) (50 mg, 0.23 mmol), EDC (53 mg,0.27 mmol), and HOBt (37 mg, 0.27 mmol) in DMSO (1 ml). The reactionmixture was stirred at room temperature for 24 hours. The reactionmixture was added dropwise to water (30 ml) and the resultant solid wascollected by filtration, washed with water and sucked dry. The titlecompound was obtained as a beige solid (15 mg, 19%). (LC/MS: R_(t) 4.08,[M+H]⁺ 344.67).

Example 49 Synthesis of Quinoline-2-carboxylicacid[3-(4-fluoro-phenylcarbamoyl)-1H-pyrazol-4-yl]-amide

The experiment was carried out in a manner analogous to that of Example48 using quinaldic acid (44 mg, 0.25 mmol) as starting acid. The productwas isolated as a brown solid (16 mg, 19%). (LC/MS: R_(t) 4.29, [M+H]⁺375.66).

Example 50 Synthesis of4-[(Thiophene-3-carbonyl)-amino]-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The experiment was carried out in a manner analogous to that of Example48 using thiophene-3-carboxylic acid (33 mg, 0.25 mmol) as startingacid. The product was isolated as a beige solid (15 mg, 20%). (LC/MS:R_(t) 3.77, [M+H]⁺ 330.61).

Example 51 4-(2-fluoro-3-methoxy-benzoylamino)-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide

2-Fluoro-3-methoxybenzoic acid (0.047 g, 0.28 mmol),4-amino-1H-pyrazole-3-carboxylic acid (4-fluoro-phenyl)-amide (Example2B) (0.055 g, 0.25 mmol), EDC (0.58 g, 0.30 mmol) and HOBt (0.041 g,0.30 mmol) were stirred at room temperature in DMSO (1.25 ml) for 5hours. The reaction mixture was poured into water (30 ml) and theresultant solid was collected by filtration and dried in a vacuum ovento give the title compound as a grey solid (0.058 g, 63%). (LC/MS: R_(t)3.99, [MH]⁺ 372.98).

Example 52 Synthesis of4-[2-(2-Pyrrolidin-1-yl-ethoxy)-benzoylamino]-1H-pyrazole-3-carboxylicacid 4-fluorophenylamide 52A 2-(2-Pyrrolidin-1-yl-ethoxy)-benzoic acidmethyl ester

Diisopropylazodicarboxylate (0.404 g, 2 mmol) was added dropwise to asolution of triphenylphosphine (0.524 g, 2 mmol) in THF (10 ml). Methylsalicylate (0.304 g, 2 mmol) was added dropwise and the resultantmixture was stirred at room temperature for 1 hour. 1,2-Hydroxyethylpyrrolidine (0.230 g, 2 mmol) was added dropwise and the reactionmixture was left stirring at room temperature for a further 1.5 hours.The resulting solution was reduced in vacuo and subject to flash columnchromatography, eluting with hexane: ethyl acetate (5:1, 1:1) then ethylacetate : methanol (4:1) to give the product as a clear yellow oil(0.104 g, 21%). (LC/MS: R_(t) 0.69, 1.62, [MH]⁺ 250.02).

52B.4-[2-(2-Pyrrolidin-1-yl-ethoxy)-benzoylamino]-1H-pyrazole-3-carboxylicacid 4-fluorophenylamide

2-(2-Pyrrolidin-1-yl-ethoxy)-benzoic acid methyl ester (0.104 g, 0.42mmol) was treated with 2 M aqueous NaOH (20 ml) and water (20 ml). Thereaction mixture was stirred at room temperature for 20 hours, thenreduced in vacuo and azeotroped with toluene (3×5 ml). Water (50 ml) wasadded and the mixture taken to pH 5 using 1M aqueous HCl. The resultingsolution was reduced in vacuo and azeotroped with toluene (3×5 ml) togive a white solid, which was combined with4-amino-1H-pyrazole-3-carboxylic acid (4-fluoro-phenyl)-amide (Example2B) (0.055 g, 0.25 mmol), EDC (0.058 g, 0.3 mmol) and HOBt (0.041g, 0.3mmol) and stirred at room temperature in DMSO (3 ml) for 20 hours. Thereaction mixture was poured into water (30 ml) and the resultant solidwas collected by filtration and dried in a vacuum oven to give the titlecompound as a grey solid (0.015 g, 14%). (LC/MS: R_(t) 2.18, [MH]⁺438.06).

Example 53 Synthesis of4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(1-methyl-piperidin-4-yl)-amide

A mixture of 4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(134 mg, 0.50 mmol), 4-amino-N-methylpiperidine (50.0 μl, 0.45 mmol),EDAC (104 mg, 0.54 mmol) and HOBt (73.0 mg, 0.54 mmol) in DMF (3 ml) wasstirred at ambient temperature for 16 hours. The mixture was reduced invacuo, the residue taken up in EtOAc and washed successively withsaturated aqueous sodium bicarbonate, water and brine. The organicportion was dried (MgSO₄) and reduced in vacuo to give4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid(1-methyl-piperidin-4-yl)-amide as a white solid (113 mg, 69%). (LC/MS:R_(t) 2.52, [M+H]⁺ 364.19).

Example 54 Synthesis of4-(Cyclohexyl-methyl-amino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

This compound was prepared in a manner analogous to the compound ofExample 19 by succssive reductive alkylations using firstlycyclohexanone and then formaldehyde. (LC/MS: R_(t) 2.77 [M H]⁺ 316.71).

Example 55 4-(Pyridin-2-ylamino)-1H-pyrazole-3-carboxylic acid(4-fluoro-phenyl)-amide

The title compound was prepared in a manner analogous to the compound ofExample 23. (LC/MS: R_(t) 2.07 [MH]⁺ 298.03).

Examples 56-81

By following the procedures described in the foregoing examples ormethods analogous thereto, or by carrying out chemical transformationsusing the compounds described in the above examples and syntheticmethods well known to the skilled person, the compounds set out in Table3 were prepared.

TABLE 3 Prepared using method analogous to Differences to Example No.Structure Example No Example? LCMS 56

 4 R_(t) 3.20 min [M + H]⁺ 406.07 57

 4 Then removal of t-Boc protecting group with TFA as described inExample 82 R_(t) 2.35 min m/z 343.72 58

 4 Used DMSO instead of DMF as solvent R_(t) 3.51 min m/z 314.62 59

 4 Used DMSO instead of DMF as solvent R_(t) 3.79 min m/z 363.67 60

48 Purified by column chromatography using EtOAC: Petroleum ether eluentR_(t) 3.68 min m/z 384.69 61

48 Purified by column chromatography using EtOAC: Petroleum ether eluentR_(t) 3.61 min m/z 326.10 62

48 Purified by column chromatography using EtOAC: Petroleum ether eluentR_(t) 3.51 min m/z 387.11 63

48 R_(t) 3.11 min m/z 313.65 64

48 Purified by column chromatography using EtOAC: Petroleum ether eluentR_(t) 2.20 min m/z 455.19 65

53 R_(t) 3.95 min m/z 349.09 66

48 Purified by column chromatography using EtOAC: Petroleum ether eluentR_(t) 2.39 min m/z 351.07 67

48 Purified by column chromatography using EtOAC: Petroleum ether eluentR_(t) 2.83 min m/z 365.13 68

Removal of PMB group from the compound of Example 62 using TFA- anisoleR_(t) 2.10 min m/z 266.97 69

48 Used DMF instead of DMSO as solvent R_(t) 3.22 min m/z 363.10 70

48 R_(t) 4.48 min m/z 358.96 71

48 R_(t) 3.93 min m/z 340.96 72

48 R_(t) 4.11 min m/z 373.01 73

48 Used DMF instead of DMSO as solvent R_(t) 2.56 min m/z 373.05 74

Obtained by oxidation and then reductive amination of Example 73 R_(t)1.99 min m/z 442.09 75

53 Purified by column chromatography using DCM:MeOH (1:0 to 19:1) eluentR_(t) 3.65 min m/z 335.03 76

25 Purified by column chromatography. Then removal of t-Boc protectinggroup with saturated ethyl acetate/HCl R_(t) 1.57 min m/z 350.10 77

53 R_(t) 5.05 min m/z 405.14 78

53 R_(t) 2.87 min m/z 416.07 79

53 Purified by column chromatography using EtOAC: Petroleum ether eluent(1:1) R_(t) 3.41 min m/z 321.03 80

2A, 2B & 53 Commercially available 5- methyl-pyrazole- 1H-3-carboxylicacid used as starting material. Purified by column chromatography usingEtOAc: Hexane eluent (1:3 to 1:1) R_(t) 3.42 min m/z 375.05 81

2C Purified by column chromatography using EtOAC: Hexane eluent (1:1 to1:0) R_(t) 2.37 min m/z 277.04

Example 824-[(4-Amino-1-methyl-1H-imidazole-2-carbonyl)-amino]-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide

Trifluoroacetic acid (200 μl) was added to a stirred suspension of{2-[3-(4-fluoro-phenylcarbamoyl)-1H-pyrazol-4-ylcarbamoyl]-1-methyl-1H-imidazol-4-yl}-carbamicacid tert-butyl ester (30 mg) in dichloromethane (5 ml), then stirred atroom temperature for 2 hours. The solvent was evaporated thenre-evaporated with toluene (2×10 ml). The residue was triturated withdiethyl ether and the resultant solid collected by filtration. The solidwas washed with diethyl ether then dried under vacuum to give 15 mg of4-[(4-amino-1-methyl-1H-imidazole-2-carbonyl)-amino]-1H-pyrazole-3-carboxylicacid (4-fluoro-phenyl)-amide as an off-white solid. (LC/MS: [M+H]⁺343.72).

Example 83 Synthesis of4-{[4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-cyclohexanecarboxylicacid 83A.4-{[4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-cyclohexanecarboxylacid ethyl ester

Thionyl chloride (0.32 ml, 4.40 mmol) was slowly added to a mixture of4-aminocyclohexanecarboxylic acid (572 mg, 4.00 mmol) in EtOH (10 ml)and stirred at ambient temperature for 16 hours. The mixture was reducedin vacuo, azeotroping with toluene, to give the corresponding ethylester (650 mg) as a pale solid.

A mixture of the ethyl ester (103 mg, 0.60 mmol),4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid (134 mg,0.50 mmol), EDC (115 mg, 0.60 mmol) and HOBt (81 mg, 0.60 mmol) in DMF(5 ml) was stirred at ambient temperature for 16 hours. The mixture wasreduced in vacuo, the residue taken up in EtOAc and washed successivelywith saturated aqueous sodium bicarbonate, water and brine. The organicportion was dried (MgSO₄) and reduced in vacuo to give4-{[4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-cyclohexanecarboxylicacid ethyl ester (112 mg).

83B.4-{[4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-cyclohexanecarboxylicacid

A mixture of the ester (45 mg) (from 83A) in MeOH (2.5 ml) and 2Maqueous NaOH (2.5 ml) was stirred at ambient temperature for 16 hours.The volatiles were removed in vacuo, water (10 ml) added and the mixturetaken to pH 5 using 1M aqueous HCl. The precipitate formed was collectedby filtration and purified by column chromatography using EtOAc/MeOH(1:0-9:1) to give4-{[4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-cyclohexanecarboxylicacid (11 mg) as a white solid and mixture of cis-/trans-isomers.

(LC/MS: R_(t) 2.78 and 2.96, [M+H]⁺ 393.09).

Examples 84-152

General Procedure A

Preparation of Amide from Pyrazole Carboxylic Acid

A mixture of the appropriate benzoylamino-1H-pyrazole-3-carboxylic acid(0.50 mmol), EDAC (104 mg, 0.54 mmol), HOBt (73.0 mg, 0.54 mmol) and thecorresponding amine (0.45 mmol) in DMF (3 ml) was stirred at ambienttemperature for 16 hours. The mixture was reduced in vacuo, the residuetaken up in EtOAc and washed successively with saturated aqueous sodiumbicarbonate, water and brine. The organic portion was dried (MgSO₄) andreduced in vacuo to give the desired product.

General Procedure B

Preparation of Amide from Amino-Pyrazole

To a stirred solution of the appropriate4-amino-1H-pyrazole-3-carboxylic acid amide (0.23 mmol), EDAC (52 mg;0.27 mmol) and HOBt (37 mg; 0.27 mmol) in 5 ml of N,N-dimethylformamidewas added the corresponding carboxylic acid (0.25 mmol), and the mixturewas then left at room temperature overnight. The reaction mixture wasevaporated and the residue purified by preparative LC/MS, to give theproduct.

General Procedure C

Deprotection of Piperidine Ring Nitrogen by Removal oftert-Butoxycarbonyl Group

A product of Procedure A or Procedure B containing a piperidine groupbearing an N-tert-butoxycarbonyl (t-Boc) protecting group (40 mg) wastreated with saturated ethyl acetate/HCl, and stirred at roomtemperature for 1 hour. A solid precipitated out of the reactionmixture, which was filtered off, washed with ether, and then dried togive 25 mg product (LC/MS: [M+H]⁺ 364).

Procedure L

Preparation of Amine Starting Materials

The following method was used to prepare the following amines:

4-thiomorpholine-4-yl-cyclohexylamine;

4-(1,1-dioxo-thiomorpholine-4-yl)-cyclohexylamine;

N-(tetrahydro-pyran-4-yl)-cyclohexane-1,4-diamine;

4-(4-methyl-piperazin-1-yl)-cyclohexylamine;

1′-methyl-[1,4′]bipiperidinyl-4-ylamine; and

4-morpholin-4-yl-cyclohexylamine.

A solution of N-4-Boc-aminocyclohexanone (0.5 g, 2.3 mmol) in THF (10ml) was treated with the appropriate amine, e.g. thiomorpholine (0.236g, 2.3 mmol), and sodium triacetoxyborohydride (0.715 g, 2.76 mmol) andacetic acid (0.182 ml). The reaction was stirred overnight at roomtemperature, then diluted with CH₂Cl₂ and washed with saturated sodiumcarbonate. The organic layer was dried over MgSO₄ and evaporated to givea white solid which was used without further purification in the nextstep. The white solid was treated with with saturated HCl/EtOAc, stirredat room temperature for 1 hour, evaporated to dryness and thenre-evaporated with toluene. The resulting amines were isolated as thehydrochloride salt. (LC/MS: R_(t) 1.75, [M+H]⁺ 201).

By following General Procedures A, B, C and L, modified where stated,the compounds set out in Table 4 were prepared.

TABLE 4 Example No. Method of Preparation LCMS 84

Procedure A [M + H]⁺ 380 R_(t) 1.42 85

Procedure A [M + H]⁺ 426 R_(t) 1.93 86

Procedure A [M + H]+ 440 R_(t) 1.87 87

Procedure A [M + H]⁺ 406 R_(t) 2.78 88

Procedure A [M + H]⁺ 406 R_(t) 2.55 89

Procedure A DMSO instead of DMF [M + H]⁺ 358 R_(t) 1.98 90

Procedure A DMSO instead of DMF [M + H]⁺ 357 R_(t) 3.37 91

Procedure A DMSO instead of DMF [M + H]⁺ 391 R_(t) 3.16 92

Procedure A DMSO instead of DMF [M + H]⁺ 375 R_(t) 3.02 93

Procedure A DMSO instead of DMF [M + H]⁺ 425 R_(t) 3.27 94

Procedure A DMSO instead of DMF [M + H]⁺ 393 R^(t) 3.01 95

Procedure A DMSO instead of DMF [M + H]⁺ 365 R_(t) 2.22 96

Procedure A DMSO instead of DMF [M + H]⁺ 387 R_(t) 3.05 97

Procedure A DMSO instead of DMF [M + H]⁺ 464 R_(t) 3.17 98

Procedure C using the product of Example 97 as starting material [M +H]⁺ 364 R_(t) 1.76 99

Procedure A DMSO instead of DMF [M + H]⁺ 389 R_(t) 2.36 100

Procedure A DMSO instead of DMF [M + H]⁺ 351 R_(t) 2.55 101

Procedure A DMSO instead of DMF [M + H]⁺ 362 R_(t) 2.63 102

Procedure A DMSO instead of DMF Starting amine prepared according toProcedure L [M + H]⁺ 364 R_(t) 1.75 103

Procedure A DMSO instead of DMF [M + H]⁺ 358 R_(t) 3.2 104

Procedure A DMSO instead of DMF [M + H]⁺ 358 R_(t) 1.77 105

Procedure A DMSO instead of DMF [M + H]⁺ 344 R_(t) 2.71 106

Procedure A DMSO instead of DMF [M + H]⁺ 392 R_(t) 2.57 107

Procedure A DMSO instead of DMF [M + H]⁺ 347 R_(t) 2.8 108

Procedure A DMSO instead of DMF [M + H]⁺ 371 R_(t) 3.1 109

Procedure A Et₃N 1 equiv., DMSO instead of DMF [M + H]⁺ 404 R_(t) 2.7110

Procedure A Et₃N 2 equiv., HOAt instead of HOBt, DMSO instead of DMF[M + H]⁺ 428 R_(t) 2.63 111

Procedure Procedure A followed by Procedure C Et₃N 2 equiv., HOAtinstead of HOBt, DMSO instead of DMF [M + H]⁺ 364 R_(t) 1.75 112

Procedure A Et₃N 2 equiv., HOAt instead of HOBt, DMSO instead of DMF[M + H]⁺ 427 R_(t) 2.71 113

Procedure A HOAt instead of HOBt, DMSO instead of DMF [M + H]⁺ 363 R_(t)3.34 114

Procedure A Et₃N 2 equiv., HOAt instead of HOBt, DMSO instead of DMF[M + H]⁺ 432 R_(t) 2.63 115

Procedure A [M + H]⁺ 461 R_(t) 3.3 116

Procedure A DMSO instead of DMF, Et₃N 2 equiv Starting amine preparedaccording to Procedure L [M + H]⁺ 448 R_(t) 1.87 117

Procedure A DMSO instead of DMF, Et₃N 2 equiv Starting amine preparedaccording to Procedure L [M + H]⁺ 447 R_(t) 1.65 118

Procedure A DMSO instead of DMF, Et₃N 2 equiv Starting amine preparedaccording to Procedure L [M + H]⁺ 447 R_(t) 1.72 119

Procedure B [M + H]⁺ 462 R_(t) 2.97 120

Procedure A N-ethyl-morpholine (NEM) 2 equiv [M + H]⁺ 379 R_(t) 2.45 121

Procedure A HOAt instead of HOBt, Et₃N 2 equiv Starting amine preparedaccording to Procedure L [M + H]⁺ 450 R_(t) 1.97 122

Procedure B [M + H]⁺ 387 R_(t) 3.83 123

Procedure B [M + H]⁺ 417 R_(t) 3.65 124

Procedure A HOAt instead of HOBt, Et₃N 2 equiv [M + H]⁺ 392 R_(t) 1.85125

Procedure A HOAt instead of HOBt, Et₃N 2 equiv [M + H]⁺ 408 R_(t) 1.82126

Procedure B [M + H]⁺ 403 R_(t) 4.02 127

Procedure B [M + H]⁺ 369 R_(t) 3.78 128

Procedure B [M + H]⁺ 435 R_(t) 3.83 129

Procedure B [M + H]⁺ 405 R_(t) 3.96 130

Procedure A HOAt instead of HOBt [M + H]⁺ 512 R_(t) 3.1 131

Procedure A HOAt instead of HOBt, [M + H]⁺ 428 R_(t) 2.45 132

Procedure A HOAt instead of HOBt, Et₃N 2 equiv. Cis and trans isomersseparated after amide coupling step Starting amine prepared according toProcedure L [M + H]⁺ 482 R_(t) 1.96 133

Procedure A HOAt instead of HOBt, DMSO instead of DMF [M + H]⁺ 434 R_(t)2.3 134

Procedure B [M + H]⁺ 442 R_(t) 2.39 135

Procedure B [M + H]⁺ 458 R_(t) 2.26 136

Procedure B HOAt instead of HOBt, [M + H]⁺ 468 R_(t) 3.07 137

Procedure A Et₃N 2 equiv., HOAt instead of HOBt, [M + H]⁺ 379 R_(t) 2.6138

Procedure B [M + H]⁺ 472 R_(t) 2.40 139

Procedure A Et₃N 2 equiv., HOAt instead of HOBt, DMSO instead of DMF[M + H]⁺ 364 R_(t) 2.1 140

Procedure B followed by Procedure C [M + H]⁺ 314 R_(t) 1.78 141

Procedure B followed by Procedure C [M + H]⁺ 332 R_(t) 1.89 142

Procedure B followed by Procedure C [M + H]⁺ 362 R_(t) 1.78 143

Procedure B followed by Procedure C [M + H]⁺ 348 R_(t) 2.01 144

Procedure B followed by Procedure C [M + H]⁺ 350 R_(t) 1.97 145

Procedure B followed by Procedure C [M + H]⁺ 380 R_(t) 2.01 146

Procedure B followed by Procedure C [M + H]⁺ 395 R_(t) 1.94 147

Procedure B followed by Procedure C [M + H]⁺ 396 R_(t) 2.11 148

Procedure B followed by Procedure C HOAt instead of HOBt [M + H]⁺ 368R_(t) 1.76 149

Procedure B followed by Procedure C [M + H]⁺ 366 R_(t) 1.78 150

Procedure B followed by Procedure C [M + H]⁺ 383 R_(t) 1.87 151

Procedure B followed by Procedure C [M + H]⁺ 433 R_(t) 1.89 152

Procedure A followed by Procedure C HOAt instead of HOBt [M + H]⁺ 350R_(t) 1.76

Examples 153-165

General Procedure D

Preparation of Protected 4-Amino-pyrazol-3-yl carboxylic acid4-hydroxy-cyclohexylamide

Step D (i):

A mixture of 4-nitro-3-pyrazolecarboxylic acid (4.98 g, 31.7 mmol),trans 4-aminocyclohexanol (3.65 g, 31.7 mmol), EDAC (6.68 g, 34.8 mmol)and HOBt (4.7 g, 34.8 mmol) in DMF (120 ml) was stirred at ambienttemperature for 16 hours. The mixture was reduced in vacuo, the residuetaken up in CH₂Cl₂ and washed successively with 5% citric acid,saturated aqueous sodium bicarbonate, water and brine. The product wasfound to be mainly in the citric acid wash, which was basified andextracted with EtOAc. The organic layer was dried over MgSO₄, filteredand evaporated to give a white solid, which was triturated with CHCl₃ togive 1.95 g of 4-nitro-1H-pyrazole-3-carboxylic acid4-hydroxy-cyclohexylamide. (LC/MS: R_(t) 1.62, [M+H]⁺ 255).

Step D (ii):

Introduction of Tetrahydro-pyran-2-yl Protecting Group

A solution of 4-nitro-1H-pyrazole-3-carboxylic acid4-hydroxy-cyclohexylamide (1.95 g; 7.67 mmol) in a mix of THF (50 ml)and chloroform (100 ml), was treated with 3,4-dihydro-2H-pyran (1.54 ml,15.34 mmol) and p-toluenesulphonic acid monohydrate (100 mg). Thereaction mixture was stirred at room temperature overnight, and thenexcess pyran (0.9 ml) was added in total to bring reaction tocompletion. The reaction mixture was diluted with CH₂Cl₂ and washedsuccessively with saturated aqueous sodium bicarbonate, water and brine.The resulting solution was reduced in vacuo and subject to Biotagecolumn chromatography, eluting with hexane (2 column lengths) followedby 30% ethyl acetate: hexane (10 column lengths), 70% ethyl acetate:hexane (10 column lengths) to give 1.25 g of4-nitro-1-(tetrahydro-pyran-2-yl-1H-pyrazole-3-carboxylicacid[4-(tetrahydro-pyran-2-yloxy)-cyclohexyl]-amide. (LC/MS: R_(t) 2.97,[M+H]⁺ 423).

Step D (iii):

A solution of 4-nitro-1-(tetrahydro-pyran-2-yl)-1H-pyrazole-3-carboxylicacid[4-(tetrahydro-pyran-2-yloxy)-cyclohexyl]-amide (0.3 g; 0.71 mmol)in methanol (25 ml), was treated with 10% palladium on carbon (30 mg)then hydrogenated at room temperature and pressure overnight. Thecatalyst was removed by filtration and washed three times with methanol.The filtrate was evaporated to give 0.264 g of the required product.(LC/MS: R_(t) 2.39, [M+H]⁺ 393).

General Procedure E

Procedure for Removal of a Tetrahydropyran-2-yl Protecting Group

To a suspension of4-(2-methoxy-benzoylamino)-1-(tetrahydro-pyran-2-yl-1H-pyrazole-3-carboxylicacid[4-(tetrahydro-pyran-2-yloxy)-cyclohexyl]-amide (0.125 g, 0.23 mmol)in EtOH (10 ml) was added p-toluene sulphonic acid hydrate (90 mg, 0.46mmol). The reaction mixture was heated at 70° C. for 30 mins. Thereaction was diluted with EtOAc and washed successively with saturatedaqueous sodium bicarbonate, water and brine. The resulting solution wasreduced in vacuo to give a white solid, which contained traces ofp-toluene sulphonic acid hydrate. The solid was then taken up in EtOAcand washed with 1M NaOH and then brine. The resulting solution wasreduced in vacuo and then triturated with ether/hexane to give 10 mg ofrequired product. (LC/MS: R_(t) 2.29, [M+H]⁺ 359)

General Procedure F

Preparation of a Urea from a 4-Amino-pyrazole-3-carboxylic acid amide

To a solution of4-amino-1-(tetrahydro-pyran-2-yl-1H-pyrazole-3-carboxylicacid[4-(tetrahydro-pyran-2-yloxy)-cyclohexyl]-amide (80 mg, 0.2 mmol) intoluene (2 ml) was added phenyl isocyanate (929 mg, 0.24 mmol). Thereaction mixture was heated at 70° C. for 1 hour. The reaction wasdiluted with EtOAc and washed successively with water and brine. Theresulting solution was reduced in vacuo to give yellow oil. This wasused without further purification. (LC/MS: R_(t) 2.28, [M+H]⁺ 344).

General Procedure G

Conversion of a 4-Amino-pyrazole Group to a4-(Morpholine-4-carbonylamino)-Pyrazole Group

To a solution of4-amino-1-(tetrahydro-pyran-2-yl-1H-pyrazole-3-carboxylicacid[4-(tetrahydro-pyran-2-yloxy)-cyclohexyl]-amide (0.1 g, 0.255 mmol)in CH₂Cl₂ (5 ml) at −10° C. was added in a dropwise manner a 20%solution of phosgene in toluene. The reaction mixture was stirred at−10° C. for 15 mins and then morpholine (0.765 mmol) was added. Thereaction mixture was allowed to warm up to room temperature over 1 hourthen stirred at room temperature overnight. The reaction was dilutedwith CH₂Cl₂ and washed successively with saturated sodium bicarbonateand brine. The resulting solution was reduced in vacuo to give a yellowoil which was used without further purification. (LC/MS: R_(t)1.68,[M+H]⁺ 338).

General Procedure H

Preparation of N-Oxides

To a suspension of the compound of Example 53 (7.7 mg, 0.02 mmol) inCH₂Cl₂ (0.5 ml) was added meta-chloroperbenzoic acid (MCPBA) (3.6 mg,0.02 mmol). The reaction mixture was stirred at room temperatureovernight, and then evaporated. The residue was purified by preparativeLC/MS, to give 3 mg of the required product. (LC/MS: R_(t) 1.83, [M+H]⁺380)

General Procedure I

Removal of a Benzyloxycarbonyl Protecting Group

A solution of the compound of Example 130 (0.2 g; 0.39 mmol) in EtOAc(40 ml) was treated with 10% palladium on carbon (20 mg) thenhydrogenated at room temperature and pressure for 3 hours. The catalystwas removed by filtration and washed three times with EtOAc. Thefiltrate was evaporated and the residue was subjected to chromatographyusing 10% MeOH-CH₂Cl₂ then 20% MeOH—CH₂Cl₂ to give 80 mg of the requiredproduct. (LC/MS: R_(t) 1.88, [M+H]⁺ 378).

General Procedure J

Mesylation of an Amine

To a solution of the compound of Example 163 (20 mg, 0.05 mmol) in CH₃CN(3 ml) added methane-sulphonyl chloride (0.0045 ml, 0.058 mmol) followedby Hunig's Base (0.018 ml, 0.1 mmol). The reaction mixture was stirredat room temperature for 2 hours and was then evaporated down. Theresidue was purified by preparative LC/MS to give 8 mg of the requiredproduct. (LC/MS: R_(t) 2.54, [M+H]⁺ 456).

By following Procedures A to L, the compounds set out in Table 5 wereprepared.

TABLE 5 Example No. Method of Preparation LCMS 153

Procedure D followed by B then E HOAt instead of HOBt, CH₂Cl₂ instead ofDMF [M + H]⁺ 359 R_(t) 2.29 154

Procedure D followed by B then E HOAt instead of HOBt, CH₂Cl₂ instead ofDMF [M + H]⁺ 377 R_(t) 2.22 155

Procedure D followed by B then E HOAt instead of HOBt, CH₂Cl₂ instead ofDMF [M + H]⁺ 381 R_(t) 2.34 156

Procedure D followed by F then E [M + H]⁺ 344 R_(t) 2.28 157

Procedure D followed by F then E [M + H]⁺ 358 R_(t) 2.22 158

Procedure D followed by B then E HOAt instead of HOBt, CH₂Cl₂ instead ofDMF [M + H]⁺ 365 R_(t) 2.21 159

Procedure D followed by B then E HOAt instead of HOBt, CH₂Cl₂ instead ofDMF [M + H]⁺ 387 R_(t) 2.29 160

Procedure D followed by F then E [M + H]⁺ 380 R_(t) 2.17 161

Procedure D followed by G then E [M + H]⁺ 338 R_(t) 1.68 162

Procedure H [M + H]⁺ 380 R_(t) 1.83 163

Procedure A (HOAt instead of HOBt) to give the compound of Example 130followed by Procedure I. [M + H]⁺ 378 R_(t) 1.78 164

Procedures A (HOAt instead of HOBt) and I to give the compound ofExample 163 followed by Procedure J. [M + H]⁺ 456 R_(t) 2.54

General Procedure M

Formation of pyrazole 4-amide Group

4-Nitropyrazole-3-carboxylic acid (7.3 g; 15.9 mmol) was added to astirred solution of 4-amino-1-Boc-piperidine (10.2 mg; 51 mmol), EDC(10.7 g; 55.8 mmol), and HOAt (55.8 g; 19.1 mmol) in DMF (100 ml), andthen stirred at room temperature overnight. The solvent was removed byevaporation under reduced pressure and the residue triturated with water(250 ml). The resultant cream solid was collected by filtration, washedwith water then dried under vacuum to give 13.05 g of4-[(4-nitro-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-carboxylic acidtert-butyl ester (LC/MS: R_(t) 2.50, [M+H]⁺ 340).

4-[(4-Nitro-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-carboxylic acidtert-butyl ester (13.05 g) was dissolved in ethanol/DMF (300 ml/75 ml),treated with 10% palladium on carbon (500 mg) then hydrogenated at roomtemperature and pressure overnight. The catalyst was removed byfiltration through Celite and the filtrate evaporated and re-evaporatedwith toluene. The crude material was purified by flash columnchromatography eluting with EtOAc then 2% MeOH/EtOAc then 5% MeOH/EtOAc.Product containing fractions were combined and evaporated to give 8.78 gof 4-[(4-amino-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-carboxylicacid tert-butyl ester as a brown foam. (LC/MS: R_(t) 1.91, [M+H]⁺ 310).

To a stirred solution of4-[(4-amino-1H-pyrazole-3-carbonyl)-amino]-piperidine-1-carboxylic acidtert-butyl ester (200 mg; 0.65 mmol), EDAC (150 mg; 0.78 mmol) and HOBt(105 mg; 0.78 mmol) in 5 ml of N,N-dimethylformamide was added thecorresponding carboxylic acid (0.25 mmol), and the mixture was then leftat room temperature overnight. The reaction mixture was diluted withsaturated aqueous sodium bicarbonate solution and the product collectedby filtration and dried under vacuum. The Boc-protected compound wasdissolved in saturated HCl/EtOAc and stirred at room temperature for 3hours. The product was collected by filtration, washed with diethylether and dried under vacuum.

General Procedure N

Preparation of 1-tert-Butyl-piperidin-4-ylamine

Step N (i)

To a solution of 1-ethyl-4-oxopiperidine (25 g, 0.197 mol) in acetone(250 ml) at RT in a water bath was added methyl iodide (15.5 ml, 0.25mol) at such a rate to keep the temperature below 30° C. The mixture wasfiltered and the precipitate washed with acetone and dried to yield1-ethyl-1-methyl-4-oxopiperidinium iodide (45 g) (LC/MS: R_(t) 0.38,[M+H]⁺ 143).

Step N (ii)

To a solution of t-butylamine (78.2 ml, 0.74 mol) in toluene (400 ml)was added a solution of 1-ethyl-1-methyl-4-oxopiperidinium iodide (40 g,0.148 mol) and sodium bicarbonate (1.245 g,0.014 mol) in water (60 ml).The reaction mixture was heated at 78° C. for 6 hours and then allowedto cool to ambient temperature. The layers were separated and theaqueous layer was washed with EtOAc. The organics were combined andwashed with brine,dried (MgSO₄), filtered and reduced in vacuo to yield1-tert-butyl-4-oxopiperidine (14 g) (LC/MS: R_(t) 0.39, [M+H]⁺ 156).

Step N (iii)

A solution of 1-tert-butyl-4-oxopiperidine (3.6 g, 23.1), benzylamine(5.1 ml, 46.8 mmol), acetic acid (1.5 ml) and sodiumtriacetoxyborohydride (7.38 g, 34.8 mmol) was stirred at ambient for 2days. Reaction mixture reduced in vacuo, residue partitioned betweenaqueous K₂CO₃ and EtOAc. The organic portion was dried (Na₂SO₄),filtered and reduced in vacuo. The residue was subjected tochromatography using CH₂Cl_(2/MeOH/NH) ₄OH (87/12/1) as the eluent toyield N-benzyl-1-tert-butylpiperidin-4-amine (1.5 g) (LC/MS: R_(t) 0.45,[M+H]⁺ 247).

Step N (iv)

A solution of N-benzyl-1-tert-butylpiperidin-4-amine (1.56 g) and 10%palladium on carbon (2 g) in MeOH (250 ml) was hydrogenated in a Parrshaker at 50 psi for 16 hours. The solution was filtered and thereaction mixture reduced in vacuo, to yield1-tert-butylpiperidin-4-amine (0.64 g) (LC/MS: R_(t) 02.31, no [M+H]⁺).

Example 165 Synthesis of4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]-amide 165A.Synthesis of 4-nitro-1H-pyrazole-3-carboxylic acid ethyl ester

Thionyl chloride (2.90 ml, 39.8 mmol) was slowly added to a mixture of4-nitro-3-pyrazolecarboxylic acid (5.68 g, 36.2 mmol) in EtOH (100 ml)at ambient temperature and the mixture stirred for 48 h. The mixture wasreduced in vacuo and dried through azeotrope with toluene to afford4-nitro-1H-pyrazole-3-carboxylic acid ethyl ester as a white solid (6.42g, 96%). (¹H NMR (400 MHz, DMSO-d₆) □ 14.4 (s, 1H), 9.0 (s, 1H), 4.4 (q,2H), 1.3 (t, 3H)).

165B. Synthesis of 4-amino-1H-pyrazole-3-carboxylic acid ethyl ester

A mixture of 4-nitro-1H-pyrazole-3-carboxylic acid ethyl ester (6.40 g,34.6 mmol) and 10% Pd/C (650 mg) in EtOH (150 ml) was stirred under anatmosphere of hydrogen for 20 h. The mixture was filtered through a plugof Celite, reduced in vacuo and dried through azeotrope with toluene toafford 4-amino-1H-pyrazole-3-carboxylic acid ethyl ester as a pink solid(5.28 g, 98%). (¹H NMR (400 MHz, DMSO-d₆) □ 12.7 (s, 1H), 7.1 (s, 1H),4.8 (s, 2H), 4.3 (q, 2H), 1.3 (t, 3H)).

165C. Synthesis of4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid ethyl ester

A mixture of 2,6-difluorobenzoic acid (6.32 g, 40.0 mmol),4-amino-1H-pyrazole-3-carboxylic acid ethyl ester (5.96 g, 38.4 mmol),EDC (8.83 g, 46.1 mmol) and HOBt (6.23 g, 46.1 mmol) in DMF (100 ml) wasstirred at ambient temperature for 6 h. The mixture was reduced invacuo, water added and the solid formed collected by filtration andair-dried to give 4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid ethyl ester as the major component of a mixture (15.3 g). (LC/MS:R_(t) 3.11, [M+H]⁺ 295.99).

165D. Synthesis of4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid

A mixture of 4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acidethyl ester (10.2 g) in 2 M aqueous NaOH/MeOH (1:1, 250 ml) was stirredat ambient temperature for 14 h. Volatile materials were removed invacuo, water (300 ml) added and the mixture taken to pH 5 using 1Maqueous HCl. The resultant precipitate was collected by filtration anddried through azeotrope with toluene to afford4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid as a pinksolid (5.70 g). (LC/MS: R_(t) 2.33, [M+H]⁺ 267.96).

165E. Synthesis of 5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenylamine

3,4-Dinitrofluorobenzene (1.86 g, 10 mmol) and4-hydroxy-1-methylpiperidine (1.38 g, 12 mmol) were dissolved in THF (20ml) and stirred at ambient temperature while sodium hydride (60%dispersion in mineral oil, 0.40 g, 10 mmol) was added in several smallportions. The reaction mixture was stirred for one hour and then reducedin vacuo, partitioned between ethyl acetate and water, and the organicphase washed with brine, dried (MgSO₄) and reduced in vacuo. Theresulting residue was subject to column chromatography, eluting with 5%MeOH/DCM to give a yellow solid (1.76 g, 2:1 ratio of4-(3,4-dinitro-phenoxy)-1-methyl-piperidine and a4-(4-fluoro-2-nitro-phenoxy)-1-methyl-piperidine).

A sample of the mixture of products obtained (0.562 g) was dissolved inDMF (10 ml) under an atmosphere of nitrogen. Palladium on carbon (10%,0.056 g) was added and the reaction mixture was shaken under a hydrogenatmosphere for 40 hours. The solids were removed by filtration and thefiltrate reduced in vacuo, taken up in ethyl acetate, washed (saturatedaqueous ammonium chloride solution, then saturated aqueous brine), dried(MgSO₄) and reduced in vacuo to give5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenylamine) as a brown oil(0.049 g, 7%). (¹H NMR (400 MHz, MeOD-d4) □ 6.6 (m, 2H), 6.4 (m, 1H),4.3 (m, 1H), 2.7 (m, 2H), 2.3 (m, 2H), 1.9 (m, 2H), 1.7 (m, 2H)).

165F. Synthesis of4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]-amide

5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenylamine) (0.049 g, 0.22mmol) was combined with4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid (0.053 g,0.20 mmol), EDC (0.048 g, 0.25 mmol), HOBt (0.034 g, 0.25 mmol) and DMF(1 ml) and the resulting reaction mixture was stirred at ambienttemperature for 18 hours. The reaction mixture was reduced in vacuo andpurified by preparative LC/MS to give4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]amide as a buffsolid. (0.010 g, 11%) (LC/MS: R_(t) 2.19, [M+H]⁺ 474.27).

Example 166 Synthesis of4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[5-fluoro-2-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amide

3,4-Dinitrofluorobenzene (0.93 g, 5 mmol) and1-(2-hydroxyethylpyrrolidine) (0.69 g, 6 mmol) were dissolved in THF (10ml) and stirred at ambient temperature while sodium hydride (60%dispersion in mineral oil, 0.24 g, 6 mmol) was added in several smallportions. The reaction mixture was stirred for 5 hours, diluted withethyl acetate and the combined organics washed with water and brine,dried (MgSO₄) and reduced in vacuo. The resulting residue was subject tocolumn chromatography, eluting with 5% MeOH/DCM to give an orange oil(0.94 g, 1:1 ratio of 1-[2-(3,4-dinitro-phenoxy)-ethyl]-pyrrolidine and1-[2-(4-Fluoro-2-nitro-phenoxy)-ethyl]-pyrrolidine.

A sample of the mixture of products obtained (0.281 g) was dissolved inDMF (5 ml) under an atmosphere of nitrogen. Palladium on carbon (10%,0.028 g) was added and the reaction mixture was shaken under a hydrogenatmosphere for 20 hours. The solids were removed by filtration and thefiltrate reduced in vacuo and combined with4-(2,6-difluoro-benzoylamino)-1H-pyrazole-3-carboxylic acid (0.134 g,0.50 mmol), EDC (0.116 g, 0.60 mmol), HOBt (0.081 g, 0.60 mmol) and DMF(2.5 ml) and the resulting reaction mixture was stirred at ambienttemperature for 18 hours. The reaction mixture was reduced in vacuo andthe residue partitioned between ethyl acetate (50 ml) and saturatedaqueous sodium bicarbonate solution (50 ml). The organic layer waswashed with brine, dried (MgSO₄) and reduced in vacuo to give theintermediate amides. Acetic acid (10 ml) was added to the crude amideand the mixture was heated at reflux for 3 hours and then reduced invacuo. 4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[5-fluoro-2-(2-pyrrolidin-1-yl-ethoxy)-phenyl]amide was isolatedfrom the residue by preparative LC/MS as an off white solid (0.040 g,5.6%). (LC/MS: R_(t) 2.38, [M+H]⁺ 474.33).

Examples 167-223

By following the procedures described above, the compounds set out inTable 6 were prepared.

TABLE 6 Example No. Structure Method Differences LCMS 167

A Starting amine prepared according to Procedure L HOAt instead of HOBtDMSO as solvent instead of DMF Et₃N 2 eq Purified by HPLC Cis/TransIsomers separated after amine preparation (L) [M + H]⁺ 434 R_(t) 1.97168

A Starting amine prepared according to Procedure L HOAt instead of HOBtDMSO as solvent instead of DMF Et₃N 2 eq Purified by chromatography 10%MeOH/CH₂Cl2 Cis/Trans Isomers separated after amine preparation (L) [M +H]⁺ 434 R_(t) 2.03 169

Procedure D followed by G then E [M + H]⁺ 338 R_(t) 2.28 170

A Starting amine prepared according to Procedure L DMSO as solventinstead of DMF Et₃N eq Heated 80° C. for 4 hours then RT O/N Purified byHPLC Cis/Trans isomers separated after final step [M + H]⁺ 448 R_(t)1.97 171

Procedure D followed by G then E [M + H]⁺ 365 R_(t) 0.34 172

B Purified by column chromatography (pet. ether-EtOAc (1:1)) [M + H]⁺414.13 R_(t) 3.05 173

B Purified by column chromatography (pet. ether-EtOAc (1:1)) [M + H]⁺432.12 R_(t) 3.12 174

B Purified by column chromatography (pet. ether-EtOAc (1:1)) [M + H]⁺448.06 R_(t) 3.33 175

B Purified by column chromatography (pet. ether-EtOAc (1:1)) [M + H]⁺450.08 R_(t) 3.29 176

B Purified by column chromatography (pet. ether-EtOAc (1:1)) [M + H]⁺480.05 R_(t) 3.18 177

A Starting amine prepared according to Procedure L HOAt instead of HOBtDMSO as solvent instead of DMF Et₃N 2 eq Purified by HPLC and formationof HCl salt [M + H]⁺ 447 R_(t) 2.01 178

B [M + H]⁺ 343.05 R_(t) 3.38 (polar method) 179

A Butyl-piperidin- 4-ylamine prepared by Procedure N HOAt instead ofHOBt Purified by trituration with MeOH [M + H]⁺ 406 R_(t) 1.85 180

B [M + H]⁺ 371.09 R_(t) 3.27 (polar method) 181

B [M + H]⁺ 306.06 R_(t) 1.53 182

B [M + H]⁺ 403.98 R_(t) 2.78 183

B [M + H]⁺ 345.05 R_(t) 3.03 184

B [M + H]⁺ 280.05 R_(t) 3.75 (basic method) 185

A HOAt instead of HOBt followed by EtOAc/HCl deprotection [M + H]⁺ 336R_(t) 1.67 186

A [M + H]⁺ 380.05 R_(t) 1.78 187

A [M + H]⁺ 396.02 R_(t) 1.86 188

A [M + H]⁺ 386.10 R_(t) 1.88 189

A [M + H]⁺ 342.10 R_(t) 1.95 190

M [M + H]⁺ = 344 R_(t) = 1.87 191

M [M + H]⁺ = 330 R_(t) = 1.80 192

M [M + H]⁺ = 372 R_(t) = 1.87 193

M [M + H]⁺ = 354 R_(t) = 1.77 194

M Purified by flash chromatography eluting with dichloromethane 120 ml,methanol 15, acetic acid 3 ml, water 2 ml (DMAW 120) [M + H]⁺ = 383/385R_(t) = 1.72 195

M Purified by flash chromatography eluting with DMAW 120 [M + H]⁺ =393/395 R_(t) = 1.86 196

M [M + H]⁺ = 398 R_(t) = 1.94 197

M [M + H]⁺ = 330 R_(t) = 1.80 198

M [M + H]⁺ = 358 R_(t) = 1.89 199

M [M + H]⁺ = 399 R_(t) = 1.88 200

M [M + H]⁺ = 420 R_(t) = 2.13 201

M [M + H]⁺ = 392/394 R_(t) = 1.84 202

B Purified using flash chromatography (CH₂Cl₂—MeOH— AcOH—H₂O(90:18:3:2)) [M + H]⁺ 376.14 R_(t) 1.78 203

B Purified using flash chromatography (CH₂Cl₂—MeOH— AcOH—H₂O(90:18:3:2)) [M + H]⁺ 400.17 R_(t) 2.08 204

B Purified using flash chromatography (CH₂Cl₂—MeOH— AcOH—H₂O(90:18:3:2)) [M + H]⁺ 376.15 R_(t) 1.92 205

B Purified using column chromatography (CH₂Cl₂—MeOH— AcOH—H₂O(90:18:3:2)) [M + H]⁺ 382.12 R_(t) 1.77 206

B Purified using column chromatography (CH₂Cl₂—MeOH— AcOH—H₂O(90:18:3:2)) [M + H]⁺ 388.18 R_(t) 1.73 207

A Purified by flash chromatography eluting with DMAW 120 [M + H]⁺ =397/399 R_(t) = 1.83 208

A Coupling using (S)-3- amino-1-N-BOC- piperidine. Deprotection asprocedure M. Purified using column chromatography (CH₂Cl₂—MeOH— AcOH—H₂O(90:18:3:2)) [M + H]⁺ 382.02 R_(t) 1.82 209

A [M + H]⁺ 440.22 R_(t) 1.92 210

A [M + H]⁺ 411.20 R_(t) 2.97 211

A Purified by prep. LCMS after work-up [M + H]⁺ 362.11 R_(t) 1.91 212

A Purified by prep. LCMS after work-up [M + H]⁺ 396.08 R_(t) 2.06 213

A Purified by prep. LCMS after work-up [M + H]⁺ 396.06 R_(t) 2.04 214

B The mixture was reduced in vacuo, the residue taken up in EtOAc andwashed successively with saturated aqueous sodium bicarbonate, water andbrine. The organic portion was dried (MgSO₄) and reduced in vacuo togive the desired product [M + H]⁺ 485 R_(t) 2.59 215

B The mixture was reduced in vacuo, the residue taken up in EtOAc andwashed successively with saturated aqueous sodium bicarbonate, water andbrine. The organic portion was dried (MgSO₄) and reduced in vacuo togive the desired product [M + H]⁺ 429 R_(t) 2.25 216

A Purified by flash chromatography eluting with DMAW 120 [M + H]⁺ = 376R_(t) = 1.85 217

A Purified by flash chromatography eluting with DMAW 120 [M + H]⁺ = 376R_(t) = 1.87 218

A Purified by flash chromatography eluting with 5% then 10% MeOH/DCM[M + H]⁺ = 376/378 R_(t) = 2.23 219

A Starting amine prepared according to Procedure L Purified by flashchromatography eluting with DMAW 90 [M + H]⁺ = 466/468 R_(t) = 1.98 220

A Purified by flash chromatography eluting with 5% then 10% MeOH/DCM[M + H]⁺ = 376/378 R_(t) = 2.09 221

A Starting amine prepared according to Procedure L Purified by flashchromatography eluting with DMAW 90 [M + H]⁺ = 434 R_(t) = 1.82 222

A Purified by flash chromatography eluting with 5% then 10% MeOH/DCM[M + H]⁺ = 356 R_(t) = 2.11 223

A Purified by flash chromatography eluting with 5% then 10% MeOH/DCM[M + H]⁺ = 344 R_(t) = 2.09

Example 224 4-(4-Methyl-piperazin-1-yl)-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide

Bis(2-chloroethyl)methylamine hydrochloride (97 mg; 0.5 mmol) was addedto a stirred solution of 4-amino-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide (100 mg; 0.45 mmol), tetrabutylammoniumiodide (20 mg; 0.045 mmol) and diisopropyethylamine (200 ul) 1.13 mmol)in DMF (5 ml), and the resulting mixture was heated at 200° C. (100 W)for 30 minutes in a CEM Discover™ microwave synthesiser. The DMF wasremoved under vacuum, then purified by flash column chromatography,eluting with dichloromethane/methanol/acetic acid/water (90:18:3:2).Product containing fractions were combined and evaporated, treated withHCl in ethyl acetate and then re-evaporated with toluene (2×20 ml) togive an off white solid (27 mg). (LC/MS: R_(t) 1.64, [M+H]⁺ 378).

Example 225 4-Morpholin-4-yl-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide

The compound was prepared in a manner analogous to Example 224, butusing bis(2-chloroethyl)ether in place of bis(2-chloroethyl)methylaminehydrochloride. (LC/MS: R_(t) 2.48 [M+H]⁺ 291).

Example 226 4-(2,4-Dichloro-phenyl)-1H-pyrazole-3-carboxylic acid4-(4-methyl-piperazin-1-yl)-benzylamide

226A. Preparation of 4-(2,4-dichloro-phenyl)-1H-pyrazole-3-carboxylicacid

A solution of 4-(2,4-dichloro-phenyl)-1H-pyrazole-3-carboxylic acidethyl ester (205 mg; 0.72 mmol) and lithium hydroxide monohydrate (125mg; 2.9 mmol) in 1:1 THF/water (10 ml) was heated at 60° C. overnight.The THF was removed by evaporation, the aqueous phase acidified with 1Mhydrochloric acid then extracted with ethyl acetate (20 ml). The ethylacetate layer was dried (MgSO₄), filtered and evaporated to give 200 mgof 4-(2,4-dichloro-phenyl)-1H-pyrazole-3-carboxylic acid. (LC/MS: [M+H]⁺256.85).

226B. Preparation of 4-(2,4-dichloro-phenyl)-1H-pyrazole-3-carboxylicacid 4-(4-methyl-piperazin-1-yl)-benzylamide

A solution of 4-(2,4-dichloro-phenyl)-1H-pyrazole-3-carboxylic acid (70mg; 0.27 mmol), 4-(4-methyl-piperazin-1-yl)-benzylamine (62 mg; 0.3mmol), EDAC (63 mg; 0.33 mmol) and HOBt (45 mg; 0.33 mmol) in 5 ml ofDMF was stirred at room temperature for 48 hours. The reaction wasevaporated and the residue partitioned between ethyl acetate and brine.The ethyl acetate layer was separated, dried (MgSO₄), filtered,evaporated then dried further under vacuum to give 34 mg of4-(2,4-dichloro-phenyl)-1H-pyrazole-3-carboxylic acid4-(4-methyl-piperazin-1-yl)-benzylamide. (LC/MS: R_(t) 2.42 [M+H]⁺ 444).

Example 227 4-(2,4-Dichloro-phenyl)-1H-pyrazole-3-carboxylic acid4-methylsulphamoylmethyl-benzylamide

The title compound was prepared in a manner analogous to Example 226,but using (4-aminomethyl-phenyl)-N-methyl-methanesulphonamide as thestarting material. 6 mg of product were isolated as a white solid.(LC/MS: R_(t) 3.56 [M+H]⁺ 440).

Example 228 4-Phenyl-1H-pyrazole-3-carboxylic acid amide

228A. 2-Benzylidene-but-3-yne nitrile

To a solution of benzaldehyde (2 g; 18.9 mmol) and malononitrile (1.37g; 20.7 mmol) in ethanol (40 ml) was added 5 drops of piperidine and themixture was heated at reflux overnight. The reaction was cooled,evaporated then purified by flash column chromatography eluting with 1:9ethyl acetate/hexane and the product containing fractions combined andevaporated to give 930 mg of 2-benzylidene-but-3-yne nitrile.

228B. 4-phenyl-5-trimethylsilanyl-1H-pyrazole-3-carbonitrile

n-Butyl lithium (2.7 M solution in heptane) (3.3 ml, 9 mmol) was addeddrop wise to a stirred solution of trimethylsilyl diazomethane (2 Msolution in diethyl ether) (4.5 ml, 9 mmol) in anhydrous THF (10 ml) at−78° C. under a nitrogen atmosphere, then stirred for a further 30minutes. To this was added drop wise a solution of2-benzylidene-but-3-yne nitrile (920 mg; 6 mmol) in anhydrous THF (5ml), the mixture stirred for 30 minutes at −78° C. then graduallyallowed to warm to room temperature overnight. The reaction mixture wasdiluted with ethyl acetate (30 ml) then washed with saturated ammoniumchloride solution followed by brine. The ethyl acetate layer wasseparated, dried (MgSO₄), filtered and evaporated. The crude product waspurified by flash column chromatography eluting with 1:8 then 1:4 ethylacetate/hexane and the product containing fractions combined andevaporated to give 1.0 g of4-phenyl-5-trimethylsilanyl-1H-pyrazole-3-carbonitrile.

228C. 4-phenyl-1H-pyrazole-3-carboxylic acid amide

4-Phenyl-5-trimethylsilanyl-1H-pyrazole-3-carbonitrile (500 mg; 2.1mmol) was dissolved in 1 ml of ethanol, treated with potassium hydroxide(600 mg) in water (3 ml) then heated at 150° C. (100 W) for 30 minutesthen 170° C. (100 W) for 20 minutes in a CEM Discover™ microwavesynthesiser. The reaction mixture was acidified to pH1 with concentratedhydrochloric acid, diluted with water (40 ml) then extracted with ethylacetate (2×40 ml). The combined ethyl acetate layers were separated,dried (MgSO₄), filtered and evaporated to give a 3:1 mixture of4-phenyl-1H-pyrazole-3-carboxylic acid and4-phenyl-1H-pyrazole-3-carboxylic acid amide. A 50 mg batch of the crudematerial was purified by flash column chromatography eluting with 5%methanol/dichloromethane, and the product containing fractions combinedand evaporated to give 15 mg of 4-phenyl-1H-pyrazole-3-carboxylic acidamide as a white solid. (LC/MS: R_(t) 2.15 [M+H]⁺ 188).

Example 229 4-phenyl-1H-pyrazole-3-carboxylic acid phenylamide

A solution of 4-phenyl-1H-pyrazole-3-carboxylic acid (75 mg; 0.4 mmol)(prepared according to Example 228C), aniline (45 ml; 0.48 mmol), EDAC(92 mg; 0.48 mmol) and HOBt (65 mg; 0.48 mmol) in 5 ml of DMF wasstirred at room temperature overnight. The reaction was evaporated thenpurified by flash column chromatography eluting with 1:3 then 1:2 ethylacetate/hexane. Product containing fractions were combined andevaporated to give 30 mg of 4-phenyl-1H-pyrazole-3-carboxylic acidphenylamide as a white solid. (LC/MS: R_(t) 3.12 [M+H]⁺ 264).

Example 230 4-Phenyl-1H-pyrazole-3-carboxylic acid4-(4-methyl-piperazin-1-yl)-benzylamide

The compound was prepared in a manner analogous to Example 229, butusing 4-(4-methyl-piperazin-1-yl)-benzylamine as the starting material.6 mg of product were isolated as a white solid. (LC/MS: R_(t) 2.05[M+H]⁺ 376).

Example 231 4-Phenyl-1H-pyrazole-3-carboxylicacid(6-methoxy-pyridin-3-yl) amide

The compound was prepared in a manner analogous to Example 230, butusing 3-amino-6-methoxypyridine as the amine fragment. 100 mg of productwere isolated as a pale brown solid. (LC/MS: R_(t) 3.17 [M+H]⁺ 295).

Example 232 4-(3-Benzyloxy-phenyl)-1H-pyrazole-3-carboxylic acid4-(4-methyl-piperazin-1-yl)-benzylamide

The compound was prepared in a manner analogous to Example 226. Theproduct was isolated as a white solid. (LC/MS: R_(t) 2.65 [M+H]⁺ 482).

Example 233 4-(3-Hydroxy-phenyl)-1H-pyrazole-3-carboxylic acid4-(4-methyl-piperazin-1-yl)-benzylamide

A solution of 4-(3-benzyloxy-phenyl)-1H-pyrazole-3-carboxylic acid4-(4-methyl-piperazin-1-yl)-benzylamide (25 mg; 0.05 mmol) in methanol(5 ml), was treated with 10% palladium on carbon (10 mg) thenhydrogenated at room temperature and pressure overnight. The catalystwas removed by filtration through Celite and the filtrate evaporated.Purification by preparative LC/MS gave 8 mg of the required product as acream solid. (LC/MS: R_(t) 1.67 [M+H]⁺ 392).

Example 234 4-(5-Methyl-3H-imidazol-4-yl)-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide

The compound was prepared in a manner analogous to Example 226, butusing 4-methyl-5-formylimidazole as the starting material in thecondensation step. The product (6 mg) was isolated as a white solid.(LC/MS: R_(t) 2.00 [M+H]⁺ 286).

Example 235 4-(2,5-Dimethyl-pyrrol-1-yl)-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide

A mixture of 4-amino-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide (100 mg) and Montmorillonite KSF clay (100mg) in acetonylacetone (1 ml) was heated at 120° C. (50 W) for 15minutes in a CEM discover microwave synthesiser. The reaction mixturewas diluted with 5% methanol/dichloromethane, filtered and evaporated.The crude product was purified by flash column chromatography elutingwith 1:2 ethyl acetate/hexane, and the product containing fractions werecombined and evaporated to give 65 mg of the target molecule as a palebrown solid. (LC/MS: R_(t) 3.75 [M+H]⁺ 299).

Example 236 4-(3-Hydroxymethyl-phenyl)-1H-pyrazole-3-carboxylic acidphenylamide

236A. 4-iodo-1H-pyrazole-3-carboxylic acid phenylamide

An aqueous solution of sodium nitrite (760 mg) in 2 ml of water wasadded drop wise to a stirred suspension of4-amino-1H-pyrazole-3-carboxylic acid phenylamide (2 g; 10 mmol) inconcentrated hydrochloric acid (20 ml) at 0° C., then stirred at 0° C.for a further 60 minutes. The reaction mixture was diluted with acetone(10 ml) then treated with potassium iodide (1.8 g) and copper (I) iodide(2.1 g) and stirred at room temperature for 90 minutes. The reactionmixture was diluted with brine and ethyl acetate then washed withsaturated sodium thiosulphate solution. The ethyl acetate layer wasseparated, dried (MgSO₄), filtered and evaporated to give 680 mg of4-iodo-1H-pyrazole-3-carboxylic acid phenylamide.

236B. 4-iodo-1-(4-methoxy-benzyl)-1H-pyrazole-3-carboxylic acidphenylamide

A solution of 4-iodo-1H-pyrazole-3-carboxylic acid phenylamide (670 mg;2.14 mmol) in acetonitrile (10 ml) was treated with potassium carbonate(360 mg; 2.57 mmol)) followed by 4-methoxybenzyl chloride (320 μl; 2.35mmol). The mixture was stirred at room temperature overnight thenevaporated under reduced pressure. The residue was partitioned betweenethyl acetate and brine; the ethyl acetate layer was separated, dried(MgSO₄), filtered and evaporated. The crude material was purified byflash column chromatography eluting with 1:3 ethyl acetate/hexane andthe product containing fractions combined and evaporated to give 660 mgof 4-iodo-1-(4-methoxy-benzyl)-1H-pyrazole-3-carboxylic acidphenylamide.

236C.4-(3-hydroxymethyl-phenyl)-1-(4-methoxy-benzyl)-1H-pyrazole-3-carboxylicacid phenylamide

A mixture of 4-iodo-1-(4-methoxy-benzyl)-1H-pyrazole-3-carboxylic acidphenylamide (50 mg; 0.11 mmol), bis(tri-tert-butylphosphine)palladium(12 mg), potassium carbonate (100 mg; 0.66 mmol) and3-(hydroxmethyl)benzene boronic acid (21 mg; 0.14 mmol) inethanol/toluene/water (4 ml:1 ml:1 ml) was heated at 120° C. (50 W) for15 minutes in a CEM Discover microwave synthesiser. The reaction wasevaporated and the residue partitioned between ethyl acetate and brine.The ethyl acetate layer was separated, dried (MgSO₄), filtered andevaporated and the crude material purified by flash columnchromatography eluting with 1:2 then 2:1 ethyl acetate/hexane. Productcontaining fractions were combined and evaporated to give 60 mg of4-(3-hydroxymethyl-phenyl)-1-(4-methoxy-benzyl)-1H-pyrazole-3-carboxylicacid phenylamide.

236D. 4-(3-Hydroxymethyl-phenyl)-1H-pyrazole-3-carboxylic acidphenylamide

A mixture of4-(3-hydroxymethyl-phenyl)-1-(4-methoxy-benzyl)-1H-pyrazole-3-carboxylicacid phenylamide (20 mg) and anisole (20 ml) in trifluoroacetic acid (1ml) was heated at 120° C. (50 W) for 15 minutes in a CEM Discovermicrowave synthesiser. The reaction was evaporated then purified byflash column chromatography eluting with 2:1 ethyl acetate/hexane.Product containing fractions were combined and evaporated to give 5 mgof product. (LC/MS: R_(t) 2.55 [M+H]⁺ 294).

Example 237 Preparation of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide hydrochloride 237A.4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acid

2,6-dichlorobenzoyl chloride (8.2 g; 39.05 mmol) was added cautiously toa solution of 4-amino-1H-pyrazole-3-carboxylic acid methyl ester(prepared in a manner analogous to 165B) (5 g; 35.5 mmol) andtriethylamine (5.95 ml; 42.6 mmol) in dioxan (50 ml) then stirred atroom temperature for 5 hours. The reaction mixture was filtered and thefiltrate treated with methanol (50 ml) and 2M sodium hydroxide solution(100 ml), heated at 50° C. for 4 hours, and then evaporated. 100 ml ofwater was added to the residue then acidified with concentratedhydrochloric acid. The solid was collected by filtration, washed withwater (100 ml) and sucked dry to give 10.05 g of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acid as a paleviolet solid.

237B.4-{[4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-piperidine-1-carboxylicacid tert-butyl ester

A mixture of 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acid(6.5 g, 21.6 mmol), 4-amino-1-BOC-piperidine (4.76 g, 23.8 mmol), EDC(5.0 g, 25.9 mmol) and HOBt (3.5 g, 25.9 mmol) in DMF (75 ml) wasstirred at room temperature for 20 hours. The reaction mixture wasreduced in vacuo and the residue partitioned between ethyl acetate (100ml) and saturated aqueous sodium bicarbonate solution (100 ml). Theorganic layer was washed with brine, dried (MgSO₄) and reduced in vacuo.The residue was taken up in 5% MeOH-DCM (˜30 ml). The insoluble materialwas collected by filtration and, washed with DCM and dried in vacuo togive4-{[4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-piperidine-1-carboxylicacid tert-butyl ester (5.38 g) as a white solid. The filtrate wasreduced in vacuo and the residue purified by column chromatography usinggradient elution 1:2 EtOAc/hexane to EtOAc to give further4-{[4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-piperidine-1-carboxylicacid tert-butyl ester (2.54 g) as a white solid.

237C. 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide

A solution of4-{[4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carbonyl]-amino}-piperidine-1-carboxylicacid tert-butyl ester (7.9 g) in MeOH (50 mL) and EtOAc (50 ml) wastreated with sat. HCl-EtOAc (40 mL) then stirred at r.t. overnight. Theproduct did not crystallise due to the presence of methanol, andtherefore the reaction mixture was evaporated and the residue trituratedwith EtOAc. The resulting off white solid was collected by filtration,washed with EtOAc and sucked dry on the sinter to give 6.3 g of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide as the hydrochloride salt. (LC/MS: R_(t) 5.89,[M+H]⁺ 382/384).

Example 238 4-Methanesulfonylamino-1H-pyrazole-3-carboxylicacid(4-fluoro-phenyl)-amide

A solution of 4-amino-1H-pyrazole-3-carboxylicacid(4-fluorophenyl)-amide (50 mg) (Example 2B) and methanesulphonicanhydride (45 mg) in pyridine (1 ml) was stirred at room temperatureovernight then evaporated and purified by flash column chromatographyeluting with 2:1 EtOAc/hexane. Evaporation of product containingfractions gave 20 mg of the title compound. (LC/MS: R_(t) 2.87; [M+H+]299).

Examples 239 to 245

The compounds of Examples 239 to 245 were prepared using the methodsdescribed above or methods closely analogous thereto.

Example 239 4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[1-(2-fluoro-ethyl)-piperidin-4-yl]-amide

Example 240 4-(2,6-Dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid(6-chloro-pyridin-3-yl)-amide

Example 241 4-(2,6-Dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid(6-amino-pyridin-3-yl)-amide

Example 242 4-(2,6-Dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid(6-methoxy-pyridin-3-yl)-amide

Example 2434-[3-Chloro-5-(4-methyl-piperazin-1-yl)-benzoylamino]-1H-pyrazole-3-carboxylicacid cyclohexylamide

Example 244 4-(2,6-Difluoro-benzoylamino)-1H-pyrazole-3-carboxylicacid[1-(2,2-difluoro-ethyl)-piperidin-4-yl]-amide

Example 2454-[3-(4-Methyl-piperazin-1-yl)-benzoylamino]-1H-pyrazole-3-carboxylicacid cyclohexylamide

Example 246 Preparation of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide acetic acid salt

To a solution of 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid piperidin-4-ylamide hydrochloride salt (Example (237C) 20.6 g, 50mmol) in water (500 ml) stirring at ambient temperature was added sodiumbicarbonate (4.5 g, 53.5 mmol). The mixture was stirred for 1 hour andthe solid formed collected by filtration and dried in vacuo azeotropingwith toluene (×3) to give the corresponding free base of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide.

¹H NMR (400 MHz, DMSO-d₆) □ 10.20 (s, 1H), 8.30 (s, 1H), 8.25 (d, 1H),7.60-7.50 (m, 3H), 3.70 (m, 1H), 3.00 (d, 2H), 2.50 (m, 2H), 1.70 (d,2H), 1.50 (m, 2H).

To a stirred suspension of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (10.0 g, 26.2 mmol) in methanol (150 ml) was addedglacial acetic acid (15 ml, 262 mmol) at ambient temperature. After 1 h,a clear solution was obtained which was reduced in vacuo azeotropingwith toluene (×2). The residue was then triturated with acetonitrile(2×100 ml) and the solid dried in vacuo to give4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide acetic acid salt (10.3 g) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) □ 10.20 (s, 1H), 8.40 (d, 1H), 8.35 (s, 1H),7.60-7.50 (m, 3H), 3.85 (m, 1H), 3.00 (d, 2H), 2.60 (t, 2H), 1.85 (s,3H), 1.70 (d, 2H), 1.55 (m, 2H)

Example 247 Synthesis of the methanesulphonic acid salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide

The methane sulphonic acid salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide may be prepared by the synthetic route shown in theScheme below.

Stage 1: Preparation of 4-nitro-1H-pyrazole-3-carboxylic acid methylester

A 20 L reaction vessel equipped with a digital thermometer and stirrerwas charged with 4-nitro-1H-pyrazole-3-carboxylic acid (1.117 Kg, 7.11mol, 1 wt) and methanol (8.950 L, 8 vol). The reaction mixture wasstirred under nitrogen, cooled to 0 to 5° C., thionyl chloride (0.581 L,8.0 mol, 0.52 vol) added over 180 minutes and the resultant mixtureallowed to warm to and stir at 18 to 22° C. overnight, after which time¹H NMR analysis (d₆-DMSO) indicated reaction completion. The reactionmixture was concentrated under reduced pressure at 40 to 45° C., theresidue treated with toluene and re-concentrated (3×2.250 L, 3×2 vol)under reduced pressure at 40 to 45° C. to give4-nitro-1H-pyrazole-3-carboxylic acid methyl ester as an off-white solid(1.210 Kg, 99.5%).

Stage 2: Preparation of 4-amino-1H-pyrazole-3-carboxylic acid methylester

A 20 L reaction vessel equipped with a digital thermometer and stirrerwas charged with palladium on carbon (10% wet paste, 0.170 Kg, 0.14 wt)under nitrogen. In a separate vessel a slurry of4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (1.210 Kg, 7.07 mol,1 wt) in ethanol (12.10 L, 10 vol) was warmed to 30 to 35° C. to effectdissolution and the solution added to the catalyst under nitrogen.Following a nitrogen-hydrogen purge sequence an atmosphere of hydrogenwas introduced and the reaction mixture maintained at 28 to 30° C. untilreaction completion (5 to 10 hours) was noted by ¹H NMR analysis(d₆-DMSO). Following a purge cycle, the reaction mixture under nitrogenwas filtered and the liquors concentrated under reduced pressure to give4-amino-1H-pyrazole-3-carboxylic acid methyl ester (0.987 Kg, 98.9%).

Stage 3: Preparation of4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid methyl ester

A solution of 4-amino-1H-pyrazole-3-carboxylic acid methyl ester (0.634Kg, 4.49 mol, 1 wt) in 1,4-dioxane (8.90 L, 9 vol) under nitrogen wastreated with triethylamine (0.761 L, 5.46 mol, 1.2 vol) followed by2,6-dichlorobenzoyl chloride (0.710 L, 4.96 mol, 0.72 vol) such that theinternal temperature was maintained in the range 20 to 25° C. Residual2,6-dichlorobenzoyl chloride was washed in with a line rinse of1,4-dioxane (0.990 L, 1 vol) and the reaction mixture stirred at 18 to25° C. until complete (16 hours) by TLC analysis (eluent: ethylacetate:heptanes 3:1; R_(f amine) 0.25, R_(f product) 0.65). Thereaction mixture was filtered, the filter-cake washed with 1,4-dioxane(2×0.990 L, 2×1 vol) and the combined filtrates (red) progressed toStage 4 without further isolation.

Stage 4: Preparation of4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid

To a solution of sodium hydroxide (0.484 Kg, 12.1 mol) in water (6.05 L)was charged a solution of the Stage 3 ester in one portion: (1.099 Kg,3.50 mol in 6.00 L). The reaction mixture was stirred to completion at20 to 25° C. as determined by TLC analysis (eluent: ethylacetate:heptanes 3:1; R_(f ester) 0.65, R_(f stage) 4 baseline). Thereaction mixture was concentrated under reduced pressure at 45 to 50°C., the oily residue diluted with water (9.90 L) and acidified to pH 1with concentrated hydrochloric acid such that the temperature wasmaintained below 30° C. The resulting precipitate was collected byfiltration, washed with water (5.00 L), pulled dry on the filter andsubsequently washed with heptanes (5.00 L). The filter-cake was chargedto a 20 L rotary evaporator flask and drying completed azeotropicallywith toluene (2×4.50 L) to afford4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid as a yellowsolid (1.044 Kg, approx. 99.5%).

Stage 5: Preparation of4-{[4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carbonyl]amino}piperidine-1-carboxylicacid tert-butyl ester

Stage 4 product (1.0 wt) and toluene (10.0 vol) were charged to asuitably sized flange flask equipped with a mechanical stirrer, droppingfunnel and thermometer. The contents were stirred under nitrogen at 16to 25° C. and thionyl chloride (0.3 vol) was added slowly. The contentswere then heated to 80 to 100° C. and stirred at this temperature untilthe reaction was judged complete by ¹H NMR. Further toluene (up to 10vol) could be added at this stage if the contents were to become toothick to stir. Once complete, the mixture was cooled to between 40 and50° C. and then concentrated under vacuum at 45 to 50° C. to dryness.The residue was then azeo-dried with toluene (3×2.0 vol).

The isolated solid was transferred to a suitably sized flask andtetrahydrofuran (5.0 vol) was charged. The contents were stirred undernitrogen at 16 to 25° C. and triethylamine (0.512 vol) was added. To aseparate flask was charged 4-amino-piperidine-1-carboxylic acidtert-butyl ester (0.704 wt) and tetrahydrofuran (5.0 vol). The contentswere agitated until complete dissolution was achieved and the solutionwas then charged to the reaction flask, maintaining the temperaturebetween 16 and 30° C. The reaction mixture was then heated to between 45and 50° C. and the contents stirred until judged complete by ¹H NMR. Thecontents were then cooled to between 16 and 25° C. and water (5.0 vol)was charged. Mixed heptanes (0.5 vol) were added, the contents werestirred for up to 10 minutes and the layers were separated. The aqueousphase was then extracted with tetrahydrofuran:mixed heptanes [(9:1),3×5.0 vol]. The organic phases were combined, washed with water (2.5vol) and then concentrated under vacuum at 40 to 45° C. The residue wasazeotroped with toluene (3×5.0 vol) and concentrated to dryness to yieldthe crude Stage 5 product.

The solid was then transferred to a suitably sized flask,methanol:toluene [(2.5:97.5), 5.0 vol] was added and the slurry wasstirred under nitrogen for 3 to 18 hours. The contents were filtered,the filter-cake was washed with toluene (2×0.7 vol) and the solid wasthen dried under vacuum at 40 to 50° C. to yield4-{[4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carbonyl]amino}piperidine-1-carboxylicacid tert-butyl ester as an off-white solid.

Two batches of Stage 4 product (0.831 kg per batch) were processed inthis way to give a total of 2.366 kg (88.6% yield) of4-{[4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carbonyl]amino}piperidine-1-carboxylicacid tert-butyl ester.

Stage 6: Preparation of4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate

Stage 5 product (1.0 wt) and 1,4-dioxane (30.0 vol) were charged to asuitably sized flange flask equipped with a mechanical stirrer, droppingfunnel and thermometer. The contents were stirred under nitrogen andheated to between 80 and 90° C. Methanesulphonic acid (0.54 vol) wasadded over 30 to 60 minutes and the contents were then heated to 95 to105° C. and stirred in this temperature range until the reaction wasjudged complete by ¹H NMR. Once complete, the contents were cooled tobetween 20 and 30° C. and the resultant precipitate collected byfiltration. The filter-cake was washed with 2-propanol (2×2.0 vol) andpulled dry on the filter for 3 to 24 hours to give crude4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate as a free-flowing off-white solid(80.0 to 120.0%w/w, uncorrected for impurities or solutes).

Several batches of Stage 5 product were processed in this way and thedetails of the quantities of starting material and product for eachbatch are set out in Table 1 below.

TABLE 1 Yields from the deprotection step - Stage 6 Input (g) of(4-{[4-(2,6- Output (g) of [4-(2,6- Dichloro-benzoylamino)-1H-Dichlorobenzoyl-amino)-1H- pyrazole-3-carbonyl]amino}-pyrazole-3-carboxylic acid piperidine-1-carboxylic acidpiperidin-4-ylamide Chemical purity Batch tert-butyl ester)methanesulphonate] (HPLC % area) 1 590.0 579.6 97.88 99.1% th, 98.2% w/w2 521.0 532.7 98.09 103.1% th, 102.2% w/w 3 523.8 511.7 98.17 98.5% th,97.7% w/w 4 518.4 596.3 98.24 116.0% th, 115.0% w/w 5 563.2 600.1 98.16107.4% th, 106.6% w/w 6 563.1 565.2 98.49 101.2% th, 100.4% w/w 7 560.4553.9 98.70 99.7% th, 98.8% w/w 8 569.7 560.6 98.41 99.2% th, 98.4% w/w

Stage 6a: Recrystallisation of 4-(26-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate

The product of Stage 6 was recrystallised to ensure that any residuallevels of Boc-protected product of Stage 5 were no greater than 0.25%.Four batches of Stage 6 product were recrystallised using the followingprotocol.

Crude Stage 6 product and 2-propanol (10.0 vol) were charged to asuitably sized flask equipped with a mechanical stirrer, dropping funneland thermometer. The contents were stirred under nitrogen and heated tobetween 75 and 85° C. Water (up to 2.5 vol) was then charged to thecontents until a clear solution was obtained. The contents were thencooled to between 40 and 60° C. and concentrated under vacuum at 40 to50° C. until the reaction volume was reduced by approximately 50%.2-Propanol (3.0 vol) was charged to the flask and the contents wereconcentrated at 40 to 50° C. until approximately 3.0 vol of solvent wasremoved. This process was then repeated twice more with 2-propanol(2×3.0 vol) and the water content was checked. The resultant slurry wasthen cooled to between 0 and 5° C. and stirred at this temperature for 1to 2 hours. The contents were filtered, the filter-cake was washed with2-propanol (2×1.0 vol) and then pulled dry on the filter for up to 24hours. The solid was transferred to drying trays and dried under vacuumat 45 to 50° C. to constant weight to give4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate as an off-white solid (60.0 to100.0% w/w).

The recrystallisation yields for the four batches ranged between 85.6%and 90.4% and the purities of the recrystallised product ranged from99.29% to 99.39%. A second recrystallisation increased the purity stillfurther.

The 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate produced by this route had amelting point (by DSC) of 379.8° C.

Removal of Residual Boc-Protected Product of Stage 5

In some cases, when the methanesulphonate salt was dissolved in acetatebuffer, a fine precipitate consisting of residual traces of theBoc-protected free base was observed. Several techniques may be used forremoving or preventing the formation of the precipitate, as set outbelow.

(a) Filtration

A mixture of the methanesulphonate salt in 200 mM acetate buffer wasdrawn from a vial into a 20 mL single-use syringe using a sterileneedle, and a clinical grade 0.2 μm filter (a Sartorius Minisart sterilesingle use filter unit) was then attached to the syringe. The plunger ofthe syringe was slowly depressed and the filtrate collected in a clean,clear glass vial. The content of the vial was a clear, colourlesssolution of the methanesulphonate salt free of particulate matter.

(b) Heating in Aqueous Acid

A mixture of the methanesulphonate salt and methanesulphonic acid (0.4eq.) in water (10 vol) was heated at 100° C. for 4 hours, and thencooled to 60° C. Analysis by TLC indicates that the methanesulphonatesalt was present as a single component. 2-Propanol (10 vol) was addedand the mixture cooled to 40° C. The mixture was reduced in vacuo toapproximately 10 volumes, then a further portion of 2-propanol added (10vol) and the mixture again reduced to 10 volumes. This cycle wasrepeated a further three times. The mixture was cooled in an ice-bathand the solid formed collected by filtration, washed with 2-propanol (5vol) and dried in vacuo to give the methanesulphonate salt as a white tooff-white solid.

(c) Organic-Aqueous Extractions

A mixture of the methanesulphonate salt and methanesulphonic acid (0.4eq.) in water (10 vol) was heated at 100° C. for 3 hours, and thencooled to ambient temperature. To this mixture was added THF-heptane(9:1, 10 vol) and the resultant mixture stirred vigorously to give asolution. The layers were separated and the aqueous phase washed withTHF-heptane (9:1, 2×10 vol) then ethyl acetate (2×10 vol). To theaqueous phase was added 2-propanol (10 vol) and the solution was reducedin vacuo to approximately 5 volumes, then a further portion of2-propanol added (10 vol) and the mixture again reduced to 5 volumes.This cycle was repeated a further three times. The solid formed wascollected by filtration, washed with 2-propanol (5 vol) and dried invacuo to give the methanesulphonate salt as a white to off-white solid.

(d) Chromatography

The use of chromatographic techniques may provide a route for removingnon-polar impurities from the methanesulphonate salt. It is envisagedthat the use of reverse-phase methods will be particularly useful.

Biological Activity

The biological activities of the compounds of (0), (I⁰), (I), (Ia),(Ib), (II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or(VIII) and sub-groups thereof as defined herein as inhibitors of CDKkinases, GSK-3 kinase and as inhibitors of cell growth are demonstratedby the examples set out below.

Example 248

Measurement of CDK2 Kinase Inhibitory Activity (IC₅₀)

Compounds of the invention were tested for kinase inhibitory activityusing either the following protocol or the activated CDK2/cyclin Akinase protocol described in Example 250.

1.7 μl of active CDK2/CyclinA (Upstate Biotechnology, 10 U/μl) isdiluted in assay buffer (25.0 μl of 10× strength assay buffer (200 mMMOPS pH 7.2, 250 mM (3-glycerophosphate, 50 mM EDTA, 150 mM MgCl₂),11.27 μl 10 mM ATP, 2.5 μl 1M DTT, 25 μl 100 mM sodium orthovanadate,708.53 μl H₂O), and 10 μl mixed with 10 μl of histone substrate mix (60μl bovine histone H1 (Upstate Biotechnology, 5 mg/ml), 940 μl H₂O, 35μCi γ³³P-ATP) and added to 96 well plates along with 5 μl of variousdilutions of the test compound in DMSO (up to 2.5%). The reaction isallowed to proceed for 5 hours before being stopped with an excess ofortho-phosphoric acid (30 μl at 2%).

γ³³P-ATP which remains unincorporated into the histone H1 is separatedfrom phosphorylated histone H1 on a Millipore MAPH filter plate. Thewells of the MAPH plate are wetted with 0.5% orthophosphoric acid, andthen the results of the reaction are filtered with a Millipore vacuumfiltration unit through the wells. Following filtration, the residue iswashed twice with 200 μl of 0.5% orthophosphoric acid. Once the filtershave dried, 25 μl of Microscint 20 scintillant is added, and thencounted on a Packard Topcount for 30 seconds.

The % inhibition of the CDK2 activity is calculated and plotted in orderto determine the concentration of test compound required to inhibit 50%of the CDK2 activity (IC₅₀).

By means of the protocol set out above, it was found that the compoundsof Examples 2C to 87, 89-92, 94, 96-101, 104-105, 165, 166, 224, 225,227, 229, 231, 233, 234 and 236 each have IC₅₀ values less than 20 μM orprovide at least 50% inhibition of the CDK2 activity at a concentrationof 10 μM. The compounds of Examples 88, 93, 226, 228, 230 and 235 eachhave IC₅₀ values less than 750 μM.

Example 249

CDK Selectivity Assays

Compounds of the invention are tested for kinase inhibitory activityagainst a number of different kinases using the general protocoldescribed in Example 247, but modified as set out below.

Kinases are diluted to a 10× working stock in 20 mM MOPS pH 7.0, 1 mMEDTA, 0.1% γ-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA.One unit equals the incorporation of 1 nmol of phosphate per minute into0.1 mg/ml histone H1, or CDK7 substrate peptide at 30° C. with a finalATP concentration of 100 uM.

The substrate for all the CDK assays (except CDK7) is histone H1,diluted to 10× working stock in 20 mM MOPS pH 7.4 prior to use. Thesubstrate for CDK7 is a specific peptide obtained from Upstate dilutedto 10× working stock in deionised water.

Assay Procedure for CDK1/cyclinB, CDK2/cyclinA, CDK2/cyclinE,CDK3/cyclinE, CDK5/p35, CDK6/cyclinD3:

In a final reaction volume of 25 μl, the enzyme (5-10 mU) is incubatedwith 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.1 mg/ml histone H1, 10 mMMgAcetate and [γ-³³P-ATP] (specific activity approx 500 cpm/pmol,concentration as required). The reaction is initiated by the addition ofMg²⁺ [γ-³³P-ATP]. After incubation for 40 minutes at room temperaturethe reaction is stopped by the addition of 5 μl of a 3% phosphoric acidsolution. 10 ml of the reaction is spotted onto a P30 filter mat andwashed 3 times for 5 minutes in 75 mM phosphoric acid and once inmethanol prior to drying and counting.

In the CDK3/cyclinE assay, the compound of Example 150 had an IC₅₀ ofless than 20 μM.

In the CDK5/p35 assay, the compounds of Examples 41 and 150 had an IC₅₀of less than 20 μM.

In the CDK6/cyclinD3 assay, the compound of Example 150 had an IC₅₀ ofless than 20 μM.

Assay Procedure for CDK7/cyclinH/MAT1

In a final reaction volume of 25 μl, the enzyme (5-10 mU) is incubatedwith 8 mM MOPS pH 7.0, 0.2 mM EDTA, 500 μM peptide, 10 mM MgAcetate and[γ-³³P-ATP] (specific activity approx 500 cpm/pmol, concentration asrequired). The reaction is initiated by the addition of Mg²+[γ-³³P-ATP].After incubation for 40 minutes at room temperature the reaction isstopped by the addition of 5 μl of a 3% phosphoric acid solution. 10 mlof the reaction is spotted onto a P30 filtermat and washed 3 times for 5minutes in 75 mM phosphoric acid and once in methanol prior to dryingand counting.

Example 250

A. Measurement of Activated CDK2/CyclinA Kinase Inhibitory ActivityAssay (IC₅₀)

Compounds of the invention were tested for kinase inhibitory activityusing the following protocol.

Activated CDK2/CyclinA (Brown et al, Nat. Cell Biol., 1, pp 438-443,1999; Lowe, E. D., et al Biochemistry, 41, pp 15625-15634, 2002) isdiluted to 125 pM in 2.5× strength assay buffer (50 mM MOPS pH 7.2, 62.5mM (3-glycerophosphate, 12.5 mM EDTA, 37.5 mM MgCl₂, 112.5 mM ATP, 2.5mM DTT, 2.5 mM sodium orthovanadate, 0.25 mg/ml bovine serum albumin),and 10 μl mixed with 10 μl of histone substrate mix (60 μl bovinehistone H1 (Upstate Biotechnology, 5 mg/ml), 940 μl H₂O, 35 μCiγ³³P-ATP) and added to 96 well plates along with 5 μl of variousdilutions of the test compound in DMSO (up to 2.5%). The reaction isallowed to proceed for 2 to 4 hours before being stopped with an excessof ortho-phosphoric acid (5 μl at 2%).

γ³³P-ATP which remains unincorporated into the histone H1 is separatedfrom phosphorylated histone H1 on a Millipore MAPH filter plate. Thewells of the MAPH plate are wetted with 0.5% orthophosphoric acid, andthen the results of the reaction are filtered with a Millipore vacuumfiltration unit through the wells. Following filtration, the residue iswashed twice with 200 μl of 0.5% orthophosphoric acid. Once the filtershave dried, 20 μl of Microscint 20 scintillant is added, and thencounted on a Packard Topcount for 30 seconds.

The % inhibition of the CDK2 activity is calculated and plotted in orderto determine the concentration of test compound required to inhibit 50%of the CDK2 activity (IC₅₀).

By means of the foregoing protocol, it was found that the compounds ofExamples 95, 96, 99-104, 106-121, 123-125, 130-137, 139, 142-145,147-150, 152-156, 158-160, 162-164, 167-173, 177-179, 181-182, 184-190,194, 196-204, 208-213 and 215 have IC₅₀ values less than 20 μM. Thecompounds of Examples 122, 126-129, 140, 141, 146, 157 and 161 each haveIC₅₀ values less than than 750 pM and most have IC₅₀ values of less than100 μM.

B. CDK1/CyclinB Assay.

CDK1/CyclinB assay is identical to the CDK2/CyclinA above except thatCDK1/CyclinB (Upstate Discovery) is used and the enzyme is diluted to6.25 nM.

In the CDK1 assay carried out as described above or by means of theprotocol set out in Example 240, the compounds of Examples 2C, 41, 48,53, 64, 65, 66, 73, 76, 77, 91, 95, 102, 106, 117, 123, 125, 133, 137,142, 150, 152, 154, 167, 186, 187, 189, 190, 193, 194, 196, 199,202-204, 207, 208-213, 215 AND 218-223 were found to have IC₅₀ valuesless than 20 μM, and the compounds of Examples 188 and 206, were foundto have I₅₀ values less than 100 μM.

Example 251

Assay Procedure for CDK4

Assays for CDK4 inhibitory activity were carried out by Proqinase GmbH,Freiburg, Germany using their proprietary 33PanQinase® Activity Assay.The assays were performed in 96 well FlashPlates™ (PerkinElmer). In eachcase, the reaction cocktail (50 μl final volume) is composed of; 20 μlassay buffer (final composition 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl₂, 3μM Na-orthovanadate, 1.2 mM DTT, 50 μg/ml PEG₂₀₀₀, 5 μl ATP solution(final concentration 1 μM [γ-33P]-ATP (approx 5×10⁵ cpm per well)), 5 μltest compound (in 10% DMSO), 10 μl substrate/10 μl enzyme solution(premixed). The final amounts of enzyme and substrate were as below.

Kinase Kinase ng/50 μl Substrate Substrate ng/50 μl CDK4/CycD1 50 Poly(Ala, Glu, Lys, Tyr) 500 6:2:5:1

The reaction cocktail was incubated at 30° C. for 80 minutes. Thereaction was stopped with 50 μl of 2% H₃PO₄, plates were aspirated andwashed twice with 200 μl 0.9% NaCl. Incorporation of ³³P was determinedwith a microplate scintillation counter. Background values weresubtracted from the data before calculating the residual activities foreach well. IC₅₀s were calculated using Prism 3.03.

The compound of Example 150 has an IC50 of less than 5 μM in this assay.

Example 252

Measurement of Inhibitory Activity Against Glycogen Synthase Kinase-3(GSK-3)

The activities of the compounds of the invention as inhibitors of GSK-3were determined using either Protocol A or Protocol B below.

Protocol A

Protocol A

GSK3-β (Upstate Discovery) is diluted to 7.5 nM in 25 mM MOPS, pH 7.00,25 mg/ml BSA, 0.0025% Brij-35®, 1.25% glycerol, 0.5 mM EDTA, 25 mMMgCl₂, 0.025% β-mercaptoethanol, 37.5 mM ATP and 10 μl mixed with 10 μlof substrate mix. The substrate mix is 12.5 μM phospho-glycogen synthasepeptide-2 (Upstate Discovery) in 1 ml of water with 35 μCi γ³³P-ATP.Enzyme and substrate are added to 96 well plates along with 5 μl ofvarious dilutions of the test compound in DMSO (up to 2.5%). Thereaction is allowed to proceed for 3 hours before being stopped with anexcess of ortho-phosphoric acid (5 μl at 2%). The filtration procedureis as for Activated CDK2/CyclinA assay above.

Protocol B

GSK3β (human) is diluted to a 10× working stock in 50 mM Tris pH 7.5,0.1 mM EGTA, 0.1 mM sodium vanadate, 0.1% β-mercaptoethanol, 1 mg/mlBSA. One unit equals the incorporation of 1 nmol of phosphate per minutephospho-glycogen synthase peptide 2 per minute.

In a final reaction volume of 25 μl, GSK3β (5-10 mU) is incubated with 8mM MOPS 7.0, 0.2 mM EDTA, 20 μM YRRAAVPPSPSLSRHSSPHQS(p)EDEEE (phosphoGS2 peptide), 10 mM MgAcetate and [Δ-³³P-ATP] (specific activity approx500 cpm/pmol, concentration as required). The reaction is initiated bythe addition of Mg²+[γ-³³P-ATP]. After incubation for 40 minutes at roomtemperature the reaction is stopped by the addition of 5 μl of a 3%phosphoric acid solution. 10 μl of the reaction is spotted onto a P30filter mat and washed 3 times for 5 minutes in 50 mM phosphoric acid andonce in methanol prior to drying and counting.

From the results of the GSK3-B assays carried out using either of thetwo protocols set out above, it was found that the compounds of Examples2C, 26, 48, 53, 65, 76, 77, 84, 86, 95, 102, 106, 119, 122, 123, 126,127, 128, 129, 131, 134, 135, 138, 140, 141, 142, 143, 144, 145, 146,147, 149, 150 and 151 each have IC₅₀ values of less than 10 μM.

Example 253

Anti-Proliferative Activity

The anti-proliferative activities of the combinations of the invention,as well as the individual components of the combinations, are determinedby measuring the ability of the compounds to inhibition of cell growthin a number of cell lines. Inhibition of cell growth is measured usingthe Alamar Blue assay (Nociari, M. M, Shalev, A., Benias, P., Russo, C.Journal of Immunological Methods 1998, 213, 157-167). The method isbased on the ability of viable cells to reduce resazurin to itsfluorescent product resorufin. For each proliferation assay cells areplated onto 96 well plates and allowed to recover for 16 hours prior tothe addition of inhibitor compounds for a further 72 hours. At the endof the incubation period 10% (v/v) Alamar Blue is added and incubatedfor a further 6 hours prior to determination of fluorescent product at535 nM ex/590 nM em. In the case of the non-proliferating cell assaycells are maintained at confluence for 96 hour prior to the addition ofinhibitor compounds for a further 72 hours. The number of viable cellsis determined by Alamar Blue assay as before. All cell lines areobtained from ECACC (European Collection of cell Cultures).

HCT-116 Cell Line

In assays against the human colon carcinoma cell line HCT 116 (ECACC No.91091005), the compounds of Examples 10, 25-27, 41, 44, 46, 48, 50, 52,53, 60, 62, 64-67, 69, 73-77, 79, 80, 83A, 86, 90-93, 95-98, 100-104,106, 107, 109-121, 123-125, 131-134, 136-143, 147-155, 158, 159,162-164, 166, 167, 178, 179, 185-190, 192-205, 207-215 and 218-223 haveIC₅₀ values of less than 20 μM and the compounds of Examples 2C, 3, 29,38, 39, 49, 51, 85, 89, 99, 108, 135, 160, 182, 183, 206 and 216 haveIC₅₀ values of less than 100 μM.

Example 254

The effect of the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (“Compound I”) in combination with 5FU, Gemcitibine,Paclitaxel, and Iressa (Compound II) were assessed using the followingtechnique:

IC₅₀ Shift Assay

Human colon carcinoma cell line HT29 (ECACC No. 91072201) cells wereseeded onto 96-well tissue culture plates at a concentration of 5×10³cells/well. Cells were allowed to recover overnight prior to addition ofcompound(s) or vehicle control (0.2% DMSO) as follows;

Compounds were added according to one of the following schedules;

-   -   a) Concurrent for 72 hours.    -   b) Compound I for 24 hours followed by Compound II for 48 hours.    -   c) Compound II for 24 hours followed by Compound I for 48 hours.

Following a total of 72 hours compound incubation, Alamar Blue™ wasadded to a final concentration of 10% (v/v) and incubated at 37° C. for6 hours. Fluorescent product was quantified by reading at d535/25x(excitation) and d590/20m (emission) on a Fusion Reader (Perkin Elmer).The IC₅₀ for Compound II in the presence of varying doses of Compound Iwas determined. Synergy was determined when the IC₅₀ shifted down in thepresence of sub-effective doses of Compound I. Additivity was determinedwhen the response to Compound II and Compound I together resulted in aneffect equivalent to the sum of the two compounds individually.Antagonistic effects were defined as those causing the IC₅₀ to shiftupwards, i.e. those where the response to the two compounds was lessthan the sum of the effect of the two compounds individually.

1. Gemcitibine

Combinations of Compound I and Gemcitibine were shown to be additive inthe IC₅₀ shift assay performed in A2780 cells. This effect was observedwhen compounds were added concurrently for 72 h or when Gemcitibine wasadded for 24 h followed by Compound I for a further 48 h. The dataobtained are summarised in FIGS. 1 and 2 below using the example of theIC₅₀ response curves to Gemcitibine in the presence and absence of 0.3μM Compound I added concurrently.

2. Paclitaxel

Combinations of Compound I and Paclitaxel were shown to be additive orsynergistic in the IC₅₀ shift assay dependent upon schedule. The studieswere performed in A2780 cells. Additive effects were observed whencompounds were added concurrently for 72 h and synergy when Paclitaxelwas added for 24 h followed by Compound I for a further 48 h. The dataobtained are summarised in FIGS. 3, 4, 5 and 6 below using examples ofthe IC₅₀ response curves to Paclitaxel in the presence and absence of0.3 μM Compound I.

3. 5FU

Combinations of Compound I and 5-FU were shown to be mildly synergisticin the IC₅₀ shift assay performed in A2780 cells. This effect wasobserved when compounds were added concurrently for 72 h. The dataobtained are summarised in FIGS. 7 and 8 below using the example of theIC₅₀ response curves to 5-FU in the presence and absence of 0.15 μMCompound I when added concurrently.

4. Iressa

Combinations of Compound I and Iressa were shown to be synergistic inthe IC₅₀ shift assay performed in A2780 cells. This effect was observedwhen compounds were added concurrently for 72 h. The data obtained aresummarised below using examples of the IC₅₀ response curves to Iressa inthe presence and absence of 0.2 μM Compound I. These data were confirmedin the HCT116 and SkBR3 cell lines.

Example 255

The effect of the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (“Compound I”) in combination with camptothecin wasassessed using the following technique:

1. IC₅₀ Shift Assay

Human colon carcinoma cell line HT29 (ECACC No. 91072201) cells wereseeded onto 96-well tissue culture plates at a concentration of 5×10³cells/well. Cells were allowed to recover overnight prior to addition ofcompound(s) or vehicle control (0.2% DMSO) as follows;

Compounds were added according to one of the following schedules;

-   -   a) Concurrent for 72 hours.    -   b) Compound I for 24 hours followed by camptothecin for 48        hours.    -   c) Camptothecin for 24 hours followed by Compound I for 48        hours.

Following a total of 72 hours compound incubation, Alamar Blue™ wasadded to a final concentration of 10% (v/v) and incubated at 37° C. for6 hours. Fluorescent product was quantified by reading at d535/25x(excitation) and d590/20m (emission) on a Fusion Reader (Perkin Elmer).The IC₅₀ for camptothecin in the presence of varying doses of Compound Iwas determined. Synergy was determined when the IC₅₀ shifted down in thepresence of sub-effective doses of Compound I. Additivity was determinedwhen the response to camptothecin and Compound I together resulted in aneffect equivalent to the sum of the two compounds individually.Antagonistic effects were defined as those causing the IC₅₀ to shiftupwards, i.e. those where the response to the two compounds was lessthan the sum of the effect of the two compounds individually.

Combinations of Compound I and camptothecin were shown to be additive inthe IC₅₀ shift assay performed in HT29 cells. This effect was observedwhen compounds were added concurrently for 72 hours or when camptothecinwas added for 24 hours followed by Compound I for a further 48 hours.Similar additivity was observed when Compound I was added prior tocamptothecin. The data obtained are summarised in FIGS. 11 and 12 belowusing the example of the IC₅₀ response curves to camptothecin in thepresence and absence of 0.1 μM Compound I in Schedule (b) (Compound Iadded followed by camptothecin).

Example 256

The effect of the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (“Compound I”) in combination with vinblastine wasassessed using the following technique:

1. IC₅₀ Shift Assay

Human colon carcinoma cell line HT29 (ECACC No. 91072201) cells wereseeded onto 96-well tissue culture plates at a concentration of 5×10³cells/well. Cells were allowed to recover overnight prior to addition ofcompound(s) or vehicle control (0.2% DMSO) as follows;

Compounds were added according to one of the following schedules;

-   -   d) Concurrent for 72 hours.    -   e) Compound I for 24 hours followed by vinblastine for 48 hours.    -   f) Vinblastine for 24 hours followed by Compound I for 48 hours.

Following a total of 72 hours compound incubation, Alamar Blue™ wasadded to a final concentration of 10% (v/v) and incubated at 37° C. for6 hours. Fluorescent product was quantified by reading at d535/25x(excitation) and d590/20m (emission) on a Fusion Reader (Perkin Elmer).The IC₅₀ for vinblastine in the presence of varying doses of Compound Iwas determined. Synergy was determined when the IC₅₀ shifted down in thepresence of sub-effective doses of Compound I. Additivity was determinedwhen the response to vinblastine and Compound I together resulted in aneffect equivalent to the sum of the two compounds individually.Antagonistic effects were defined as those causing the IC₅₀ to shiftupwards, i.e. those where the response to the two compounds was lessthan the sum of the effect of the two compounds individually.

Combinations of Compound I and Vinblastine were shown to be additive inthe IC₅₀ shift assay performed in A2780 cells. This effect was observedwhen compounds were added concurrently for 72 h or when Vinblastine wasadded for 24 h followed by compound I for a further 48 h. The dataobtained are summarised below in FIGS. 13 and 14 using the example ofthe IC₅₀ response curves to Vinblastine in the presence and absence of0.3 μM Compound I when added concurrently.

Example 257

The effect of the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (“Compound I”) in combination with cisplatin wasassessed using the following technique:

1. IC₅₀ Shift Assay

Human colon carcinoma cell line HT29 (ECACC No. 91072201) cells wereseeded onto 96-well tissue culture plates at a concentration of 5×10³cells/well. Cells were allowed to recover overnight prior to addition ofcompound(s) or vehicle control (0.2% DMSO) as follows;

Compounds were added according to one of the following schedules;

-   -   g) Concurrent for 72 hours.    -   h) Compound I for 24 hours followed by cisplatin for 48 hours.    -   i) Cisplatin for 24 hours followed by Compound I for 48 hours.

Following a total of 72 hours compound incubation, Alamar Blue™ wasadded to a final concentration of 10% (v/v) and incubated at 37° C. for6 hours. Fluorescent product was quantified by reading at d535/25x(excitation) and d590/20m (emission) on a Fusion Reader (Perkin Elmer).The IC₅₀ for cisplatin in the presence of varying doses of Compound Iwas determined. Synergy was determined when the IC₅₀ shifted down in thepresence of sub-effective doses of Compound I. Additivity was determinedwhen the response to cisplatin and Compound I together resulted in aneffect equivalent to the sum of the two compounds individually.Antagonistic effects were defined as those causing the IC₅₀ to shiftupwards, i.e. those where the response to the two compounds was lessthan the sum of the effect of the two compounds individually.

Combinations of compound I and Cisplatin were shown to be additive inthe IC₅₀ shift assay performed in A2780 cells. This effect was observedwhen compounds were added concurrently for 72 h or when Cisplatin wasadded for 24 h followed by compound I for a further 48 h. The dataobtained are summarised below in FIGS. 15 and 16 using the example ofthe IC₅₀ response curves to Cisplatin in the presence and absence of 0.3μM compound I when added concurrently.

Example 258

The effect of the compound4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (“Compound I”) in combination with etoposide wasassessed using the following technique:

1. IC₅₀ Shift Assay

Human colon carcinoma cell line HT29 (ECACC No. 91072201) cells wereseeded onto 96-well tissue culture plates at a concentration of 5×10³cells/well. Cells were allowed to recover overnight prior to addition ofcompound(s) or vehicle control (0.2% DMSO) as follows;

Compounds were added according to one of the following schedules;

-   -   j) Concurrent for 72 hours.    -   k) Compound I for 24 hours followed by etoposide for 48 hours.    -   l) Etoposide for 24 hours followed by Compound I for 48 hours.

Following a total of 72 hours compound incubation, Alamar Blue™ wasadded to a final concentration of 10% (v/v) and incubated at 37° C. for6 hours. Fluorescent product was quantified by reading at d535/25x(excitation) and d590/20m (emission) on a Fusion Reader (Perkin Elmer).The IC₅₀ for etoposide in the presence of varying doses of Compound Iwas determined. Synergy was determined when the IC₅₀ shifted down in thepresence of sub-effective doses of Compound I. Additivity was determinedwhen the response to etoposide and Compound I together resulted in aneffect equivalent to the sum of the two compounds individually.Antagonistic effects were defined as those causing the IC₅₀ to shiftupwards, i.e. those where the response to the two compounds was lessthan the sum of the effect of the two compounds individually.

Combinations of Compound I and Etoposide were shown to be additive inthe IC₅₀ shift assay performed in A2780 cells. This effect was observedwhen compounds were added concurrently for 72 h or when Etoposide wasadded for 24 h followed by compound I for a further 48 h. The dataobtained are summarised below in FIGS. 17 and 18 using the example ofthe IC₅₀ response curves to Etoposide in the presence and absence of0.075 μM Compound I when added concurrently.

Pharmaceutical Formulations

Example 259

i) Lyophilised Formulation I

Aliquots of formulated compound of formula (0), (I⁰), (I), (Ia), (Ib),(II), (III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof as defined herein are put into 50 mL vials andlyophilized. During lyophilisation, the compositions are frozen using aone-step freezing protocol at (−45° C.). The temperature is raised to−10° C. for annealing, then lowered to freezing at −45° C., followed byprimary drying at +25° C. for approximately 3400 minutes, followed by asecondary drying with increased steps if temperature to 50° C. Thepressure during primary and secondary drying is set at 80 millitor.

ii) Injectable Formulation II

A formulation for i.v. delivery by injection or infusion can be preparedby dissolving the compound of formula (0), (I⁰), (I), (Ia), (Ib), (II),(III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof as defined herein (e.g. in a salt form) in water at20 mg/ml. The vial is then sealed and sterilised by autoclaving.

iii) Injectable Formulation III

A formulation for i.v. delivery by injection or infusion can be preparedby dissolving the compound of formula (0), (I⁰), (I), (Ia), (Ib), (II),(III), (IV), (IVa), (Va), (Vb), (VIa), (VIb), (VII) or (VIII) andsub-groups thereof as defined herein (e.g. in a salt form) in watercontaining a buffer (e.g. 0.2 M acetate pH 4.6) at 20 mg/ml. The vial isthen sealed and sterilised by autoclaving.

iv) Injectable Formulation IV

A parenteral composition for administration by injection can be preparedby dissolving a compound of the formula (I) (e.g. in a salt form) inwater containing 10% propylene glycol to give a concentration of activecompound of 1.5% by weight. The solution is then sterilised byfiltration, filled into an ampoule and sealed.

(v) Injectable Formulation V

A parenteral composition for injection is prepared by dissolving inwater a compound of the formula (I) (e.g. in salt form) (2 mg/ml) andmannitol (50 mg/ml), sterile filtering the solution and filling intosealable 1 ml vials or ampoules.

(vi) Subcutaneous Injection Formulation VI

A composition for sub-cutaneous administration is prepared by mixing acompound of the formula (I) with pharmaceutical grade corn oil to give aconcentration of 5 mg/ml. The composition is sterilised and filled intoa suitable container.

(vii) Tablet Formulation

A tablet composition containing a compound of the formulae (I⁰) or (I)or an acid addition salt thereof as defined herein is prepared by mixing50 mg of the compound or its salt with 197 mg of lactose (BP) asdiluent, and 3 mg magnesium stearate as a lubricant and compressing toform a tablet in known manner.

(viii) Capsule Formulation

A capsule formulation is prepared by mixing 100 mg of a compound of theformulae (I⁰) or (I) or an acid addition salt thereof as defined hereinwith 100 mg lactose and filling the resulting mixture into standardopaque hard gelatin capsules.

(ix) Lyophilised Formulation

Aliquots of formulated compound of formulae (I⁰) or (I) or an acidaddition salt thereof as defined herein are put into 50 mL vials andlyophilized. During lyophilisation, the compositions are frozen using aone-step freezing protocol at (−45° C.). The temperature is raised to−10° C. for annealing, then lowered to freezing at −45° C., followed byprimary drying at +25° C. for approximately 3400 minutes, followed by asecondary drying with increased steps if temperature to 50° C. Thepressure during primary and secondary drying is set at 80 millitor.

(x) Concentrate for Use in i.v. Administration

An aqueous buffered solution is prepared by dissolving4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate at a concentration of 20 mg/ml ina 0.2M sodium acetate/acetic acid buffer at a pH of 4.6.

The buffered solution is filled, with filtration to remove particulatematter, into a container (such as class 1 glass vials) which is thensealed (e.g. by means of a Florotec stopper) and secured (e.g. with analuminium crimp). If the compound and formulation are sufficientlystable, the formulation is sterilised by autoclaving at 121° C. for asuitable period of time. If the formulation is not stable toautoclaving, it can be sterilised using a suitable filter and filledunder sterile conditions into sterile vials. For intravenousadministration, the solution can be dosed as is, or can be injected intoan infusion bag (containing a pharmaceutically acceptable excipient,such as 0.9% saline or 5% dextrose), before administration.

(xi) Injectable Formulation of a Camptothecin Compound

A parenteral pharmaceutical formulation for administration by injectionand containing a camptothecin compound can be prepared by dissolving 100mg of a water soluble salt of the camptothecin compound (for example acompound as described in EP 0321122 and in particular the examplestherein) in 10 ml of sterile 0.9% saline and then sterilising thesolution and filling the solution into a suitable container

Example 256 Determination of the Crystal Structure of4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate by X-ray Diffraction

The compound 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate was prepared as described inExample 1. The crystal used for the diffraction experiment was acolourless plate with dimensions 0.05×0.08×0.14 mm³ obtained byprecipitation from a water solution by 2-propanol. Crystallographic datawere collected at 93 K using CuKα radiation (λ=1.5418 Å) from a Rigakurotating anode RU3HR, Osmic blue confocal optics and a Rigaku JupiterCCD detector. Images were collected in two w scans at 2θ=15 and 90° witha detector to crystal distance of 67 mm. Data collection was controlledby CrystalClear software and images were processed and scaled by Dtrek.Due to a high absorption coefficient (μ=4.01 mm⁻¹) data had to becorrected using 4^(th) order Fourier absorption correction. It was foundthat the crystals belong to an orthorhombic space group Pbca (#61) withcrystal lattice parameters at 93 K a=8.90(10), b=12.44(10), c=38.49(4)Å, α=β=γ=90°. The numbers in brackets represents the deviation (s.u.,standard uncertainty).

The crystals described above and the crystal structure form a furtheraspect of the invention.

The crystal structure was solved using direct methods implemented inSHELXS-97. Intensity data for a total of 2710 unique reflections in aresolution range from 20-0.9 Å (2.3<θ<58.87) were used in the refinementof 271 crystallographic parameters by SHELXL-97. Final statisticalparameters were: wR2=0.2115 (all data), R1=0.0869 (data with I>2σ(I))and goodness of fit S=1.264.

One molecule of protonated free base and one mesylate anion were foundin the asymmetric unit. The elemental composition of the asymmetric unitwas C₁₇H₂₁Cl₂N₅O₅S and the calculated density of the crystals is 1.49Mg/m³. Hydrogen atoms were generated on geometrical grounds while thelocation of heteroatom bound hydrogen atoms was confirmed by inspectionof Fo-Fc difference maps. The positional and thermal parameters ofhydrogen atoms were constricted to ride on corresponding non-hydrogenatoms. The thermal motion of non-hydrogen atoms was modeled byanisotropical thermal factors (see FIG. 19).

The crystal structure contains one intramolecular (N15H . . . O7 2.690Å) and five intermolecular hydrogen bonds (see packing figure FIG. 20).Three of them link the protonated piperidine nitrogen with two mesylateanions. The first mesylate anion is linked through a single H-bondN12H12A . . . O2M 2.771 Å, while the second is involved in a bifurcatedH-bond with interactions N12H12B . . . O1M 2.864 Å and N12H12B . . . O2M3.057 Å. The remaining mesylate oxygen O3M is involved in a hydrogenbond N8H8 . . . O3M 2.928 Å. Neighbouring protonated free base moleculesare linked together by a H-bond N15H15 . . . O7 2.876 Å, as well as byrelatively long contact N15H15 . . . N2 3.562 Å and stacking of phenyland pyrazole rings. These interactions are propagated infinitely alongthe b axis. Crystal packing contains 2D layers (in the ab plane) ofmesylate anions sandwiched by an extensive network of charged H-bondswith two layers of protonated free base cations. The compact 2D sandwichlayers are joined together along the c axis by stacking of phenyl ringsand involving chlorine . . . phenyl interaction with C12 . . . C18 3.341Å.

A graphical representation of the structure generated by the X-raydiffraction study is provided in FIG. 20.

The coordinates for the atoms making up the structure of the4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide methanesulphonate are as set out in Table 2.

TABLE 2 space group: Pbca unit cell at 93K with a, b & c having 5% s.u.:a = 8.9 b = 12.4 c = 38.5 alpha = beta = gamma = 90 Coordinates in cifformat: loop_(—) _atom_site_label _atom_site_type_symbol_atom_site_fract_x _atom_site_fract_y _atom_site_fract_z_atom_site_U_iso_or_equiv _atom_site_adp_type _atom_site_occupancy_atom_site_symmetry_multiplicity _atom_site_calc_flag_atom_site_refinement_flags _atom_site_disorder_assembly_atom_site_disorder_group S1M S 0.13517(17) 0.18539(13) 0.03193(5)0.0286(5) Uani 1 1 d . . . O1M O 0.1193(5) 0.2208(3) −0.00409(14)0.0326(13) Uani 1 1 d . . . O2M O 0.1551(5) 0.0681(3) 0.03330(13)0.0331(13) Uani 1 1 d . . . O3M O 0.0151(5) 0.2217(4) 0.05453(14)0.0368(13) Uani 1 1 d . . . C4M C 0.3036(8) 0.2420(6) 0.0475(2)0.0355(19) Uani 1 1 d . . . H4M1 H 0.3855 0.2197 0.0329 0.053 Uiso 1 1calc R . . H4M2 H 0.3212 0.2181 0.0708 0.053 Uiso 1 1 calc R . . H4M3 H0.2959 0.3189 0.0471 0.053 Uiso 1 1 calc R . . Cl1 Cl 0.26158(17)0.18137(12) 0.34133(5) 0.0325(5) Uani 1 1 d . . . Cl2 Cl 0.75698(19)0.16766(13) 0.26161(5) 0.0366(6) Uani 1 1 d . . . N1 N 0.6277(6)−0.2419(4) 0.34903(16) 0.0276(14) Uani 1 1 d . . . H1 H 0.5932 −0.30640.3484 0.033 Uiso 1 1 calc R . . N2 N 0.7505(5) −0.2150(4) 0.36663(16)0.0286(15) Uani 1 1 d . . . C3 C 0.7635(7) −0.1082(5) 0.36163(19)0.0265(17) Uani 1 1 d . . . C4 C 0.6453(7) −0.0708(5) 0.34039(18)0.0211(16) Uani 1 1 d . . . C5 C 0.5616(7) −0.1594(5) 0.3322(2)0.0277(18) Uani 1 1 d . . . H5 H 0.4770 −0.1623 0.3181 0.033 Uiso 1 1calc R . . C6 C 0.8878(7) −0.0454(5) 0.3760(2) 0.0269(17) Uani 1 1 d . .. O7 O 0.9037(5) 0.0506(3) 0.36722(14) 0.0368(13) Uani 1 1 d . . . N8 N0.9821(6) −0.0939(4) 0.39821(15) 0.0267(14) Uani 1 1 d . . . H8 H 0.9626−0.1584 0.4048 0.032 Uiso 1 1 calc R . . C9 C 1.1147(7) −0.0417(5)0.41139(19) 0.0253(17) Uani 1 1 d . . . H9 H 1.1272 0.0261 0.3987 0.030Uiso 1 1 calc R . . C10 C 1.1019(8) −0.0148(5) 0.4502(2) 0.0330(18) Uani1 1 d . . . H10A H 1.0156 0.0315 0.4540 0.040 Uiso 1 1 calc R . . H10B H1.0866 −0.0804 0.4633 0.040 Uiso 1 1 calc R . . C11 C 1.2429(7)0.0412(5) 0.4630(2) 0.0349(19) Uani 1 1 d . . . H11A H 1.2533 0.11020.4515 0.042 Uiso 1 1 calc R . . H11B H 1.2355 0.0538 0.4878 0.042 Uiso1 1 calc R . . N12 N 1.3784(6) −0.0279(4) 0.45532(16) 0.0258(14) Uani 11 d . . . H12A H 1.4618 0.0069 0.4623 0.031 Uiso 1 1 calc R . . H12B H1.3716 −0.0892 0.4676 0.031 Uiso 1 1 calc R . . C13 C 1.3929(7)−0.0546(6) 0.4181(2) 0.0314(18) Uani 1 1 d . . . H13A H 1.4790 −0.10130.4147 0.038 Uiso 1 1 calc R . . H13B H 1.4098 0.0107 0.4049 0.038 Uiso1 1 calc R . . C14 C 1.2538(7) −0.1097(6) 0.4049(2) 0.0356(19) Uani 1 1d . . . H14A H 1.2425 −0.1785 0.4165 0.043 Uiso 1 1 calc R . . H14B H1.2639 −0.1231 0.3802 0.043 Uiso 1 1 calc R . . N15 N 0.6215(5)0.0371(4) 0.33108(16) 0.0256(14) Uani 1 1 d . . . H15 H 0.6768 0.08520.3408 0.031 Uiso 1 1 calc R . . C16 C 0.5183(7) 0.0697(5) 0.30805(18)0.0213(15) Uani 1 1 d . . . O17 O 0.4336(5) 0.0082(3) 0.29260(13)0.0309(12) Uani 1 1 d . . . C18 C 0.5120(6) 0.1890(5) 0.30170(17)0.0195(15) Uani 1 1 d . . . C19 C 0.3923(7) 0.2486(5) 0.31620(19)0.0252(16) Uani 1 1 d . . . C20 C 0.3785(7) 0.3569(5) 0.30904(19)0.0267(17) Uani 1 1 d . . . H20 H 0.2991 0.3957 0.3185 0.032 Uiso 1 1calc R . . C21 C 0.4814(7) 0.4078(5) 0.28805(19) 0.0270(17) Uani 1 1 d .. . H21 H 0.4708 0.4808 0.2834 0.032 Uiso 1 1 calc R . . C22 C 0.6005(7)0.3518(5) 0.27375(19) 0.0294(18) Uani 1 1 d . . . H22 H 0.6702 0.38650.2597 0.035 Uiso 1 1 calc R . . C23 C 0.6142(7) 0.2425(5) 0.2807(2)0.0286(17) Uani 1 1 d . . .

Example 257 Preparation of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide acetic acid salt

To a solution of 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid piperidin-4-ylamide hydrochloride salt (20.6 g, 50 mmol) in water(500 ml) stirring at ambient temperature was added sodium bicarbonate(4.5 g, 53.5 mmol). The mixture was stirred for 1 hour and the solidformed collected by filtration and dried in vacuo azeotroping withtoluene (×3) to give the corresponding free base of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide.

¹H NMR (400 MHz, DMSO-d₆) δ 10.20 (s, 1H), 8.30 (s, 1H), 8.25 (d, 1H),7.60-7.50 (m, 3H), 3.70 (m, 1H), 3.00 (d, 2H), 2.50 (m, 2H), 1.70 (d,2H), 1.50 (m, 2H).

To a stirred suspension of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide (10.0 g, 26.2 mmol) in methanol (150 ml) was addedglacial acetic acid (15 ml, 262 mmol) at ambient temperature. After 1 h,a clear solution was obtained which was reduced in vacuo azeotropingwith toluene (×2). The residue was then triturated with acetonitrile(2×100 ml) and the solid dried in vacuo to give4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide acetic acid salt (10.3 g) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.20 (s, 1H), 8.40 (d, 1H), 8.35 (s, 1H),7.60-7.50 (m, 3H), 3.85 (m, 1H), 3.00 (d, 2H), 2.60 (t, 2H), 1.85 (s,3H), 1.70 (d, 2H), 1.55 (m, 2H)

Equivalents

The foregoing examples are presented for the purpose of illustrating theinvention and should not be construed as imposing any limitation on thescope of the invention. It will readily be apparent that numerousmodifications and alterations may be made to the specific embodiments ofthe invention described above and illustrated in the examples withoutdeparting from the principles underlying the invention. All suchmodifications and alterations are intended to be embraced by thisapplication.

1.-376. (canceled)
 377. A combination comprising: (i) a cytotoxiccompound or signaling inhibitor; or (ii) an ancillary agent; or (iii)two or more further anti-cancer agents; and a compound of the formula(Ib):

or salts or tautomers or N-oxides or solvates thereof; wherein X is agroup R¹-A-NR⁴-; A is a bond, C═O, NR^(g)(C═O) or O(C═O) wherein R^(g)is hydrogen or C₁₋₄ hydrocarbyl optionally substituted by hydroxy orC₁₋₄ alkoxy; Y is a bond or an alkylene chain of 1, 2 or 3 carbon atomsin length; R¹ is a carbocyclic or heterocyclic group having from 3 to 12ring members; or a C₁₋₈ hydrocarbyl group optionally substituted by oneor more substituents selected from fluorine, hydroxy, C₁₋₄hydrocarbyloxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, andcarbocyclic or heterocyclic groups having from 3 to 12 ring members, andwherein 1 or 2 of the carbon atoms of the hydrocarbyl group mayoptionally be replaced by an atom or group selected from O, S, NH, SO,SO₂; R² is hydrogen; halogen; C₁₋₄ alkoxy (e.g. methoxy); or a C₁₋₄hydrocarbyl group optionally substituted by halogen (e.g. fluorine),hydroxyl or C₁₋₄ alkoxy (e.g. methoxy); R³ is selected from carbocyclicand heterocyclic groups having from 3 to 12 ring members; and R⁴ ishydrogen or a C₁₋₄ hydrocarbyl group optionally substituted by halogen(e.g. fluorine), hydroxyl or C₁₋₄ alkoxy (e.g. methoxy).
 378. Acombination according to claim 377 wherein R¹ is a carbocyclic orheterocyclic group having from 3 to 12 ring members which is optionallysubstituted by one or more substituent groups R¹⁰ selected from halogen,hydroxy, trifluoromethyl, cyano, nitro, carboxy, amino, mono- or di-C₁₋₄hydrocarbylamino, carbocyclic and heterocyclic groups having from 3 to12 ring members; a group R^(a)—R^(b) wherein R^(a) is a bond, O, CO,X¹C(X²), C(X²)X¹, X¹C(X²)X¹, S, SO, SO₂, NR^(c), SO₂NR^(c) or NR^(c)SO₂;and R^(b) is selected from hydrogen, carbocyclic and heterocyclic groupshaving from 3 to 12 ring members, and a C₁₋₈ hydrocarbyl groupoptionally substituted by one or more substituents selected fromhydroxy, oxo, halogen, cyano, nitro, carboxy, amino, mono- or di-C₁₋₄hydrocarbylamino, carbocyclic and heterocyclic groups having from 3 to12 ring members and wherein one or more carbon atoms of the C₁₋₈hydrocarbyl group may optionally be replaced by O, S, SO, SO₂, NR^(c),X¹C(X²), C(X²)X¹ or X¹C(X²)X¹; R^(c) is selected from hydrogen and C₁₋₄hydrocarbyl; and X¹ is O, S or NR^(c) and X² is ═O, ═S or ═NR^(c). 379.A combination according to claim 377 wherein the compound of formula(Ib) is a compound having the formula (II):


380. A combination according to claim 377 wherein the compound offormula (Ib) is a compound having the formula (IV):

or salts or tautomers or N-oxides or solvates thereof; an optionalsecond bond may be present between carbon atoms numbered 1 and 2; one ofU and T is selected from CH₂, CHR¹³, CR¹¹R¹³, NR¹⁴, N(O)R¹⁵, O andS(O)_(t); and the other of U and T is selected from, NR¹⁴, O, CH₂,CHR¹¹, C(R¹¹)₂, and C═O; r is 0, 1, 2, 3 or 4; t is 0, 1 or 2; R¹¹ isselected from hydrogen, halogen (particularly fluorine), C₁₋₃ alkyl(e.g. methyl) and C₁₋₃ alkoxy (e.g. methoxy); R¹³ is selected fromhydrogen, NHR¹⁴, NOH, NOR¹⁴ and R^(a)—R^(b); R¹⁴ is selected fromhydrogen and R^(d)—R^(b); R^(d) is selected from a bond, CO, C(X²)X¹,SO₂ and SO₂NR^(c); R^(a) is a bond, O, CO, X¹C(X²), C(X²)X¹, X¹C(X²)X¹,S, SO, SO₂, NR^(c), SO₂NR^(c) or NR^(c)SO₂; R^(b) is selected fromhydrogen, carbocyclic and heterocyclic groups having from 3 to 12 ringmembers, and a C₁₋₈ hydrocarbyl group optionally substituted by one ormore substituents selected from hydroxy, oxo, halogen, cyano, nitro,carboxy, amino, mono- or di-C₁₋₄ hydrocarbylamino, carbocyclic andheterocyclic groups having from 3 to 12 ring members and wherein one ormore carbon atoms of the C₁₋₈ hydrocarbyl group may optionally bereplaced by O, S, SO, SO₂, NR^(c), X¹C(X²), C(X²)X¹ or X¹C(X²)X¹; R^(c)is selected from hydrogen and C₁₋₄ hydrocarbyl; X¹ is O, S or NR^(c) andX² is ═O, ═S or ═NR^(c); and R¹⁵ is selected from C₁₋₄ saturatedhydrocarbyl optionally substituted by hydroxy, C₁₋₂ alkoxy, halogen or amonocyclic 5- or 6-membered carbocyclic or heterocyclic group, providedthat U and T cannot be O simultaneously.
 381. A combination according toclaim 377 wherein the compound of formula (Ib) is a compound the formula(VIb):

or salts or tautomers or N-oxides or solvates thereof; wherein R²⁰ isselected from hydrogen and methyl; R^(21a) is selected from fluorine andchlorine; and R^(22a) is selected from fluorine, chlorine and methoxy.382. A combination according to claim 381 wherein the compound of theformula (VIb) is 4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylicacid piperidin-4-ylamide or a salt thereof.
 383. A combination accordingto claim 382 wherein the salt of4-(2,6-dichloro-benzoylamino)-1H-pyrazole-3-carboxylic acidpiperidin-4-ylamide is the salt formed with methanesulphonic acid. 384.A combination according to claim 377 wherein the cytotoxic compound orsignaling inhibitor, or an ancillary agent, or two or more furtheranti-cancer agents, and compound of formula (Ib) are physicallyassociated.
 385. The combination of claim 377 wherein the cytotoxiccompound or signaling inhibitor, or an ancillary agent, or two or morefurther anti-cancer agents, and compound of formula (Ib) arenon-physically associated.
 386. A method of inhibiting tumour growth ina mammal, which method comprises administering to the mammal aneffective tumour growth-inhibiting amount of a combination according toclaim
 377. 387. A method for preventing, treating or managing cancer ina patient in need thereof, said method comprising administering to saidpatient a prophylactically or therapeutically effective amount of acombination according to claim
 377. 388. A method of enhancing orpotentiating the response rate in a patient suffering from a cancerwhere the patient is being treated with a cytotoxic compound orsignaling inhibitor, or an ancillary agent, or two or more furtheranti-cancer agents, which method comprises administering to the patient,in combination with the cytotoxic compound or signaling inhibitor, or anancillary agent, or two or more further anti-cancer agents, a compoundof Formula (Ib) as defined in claim
 377. 389. A combination according toclaim 377 wherein the cytotoxic compound is selected from camptothecincompounds; antimetabolites; vinca alkaloids; taxanes; platinumcompounds; DNA binders and Topo II inhibitors (including anthracyclinederivatives); and a combination of two or more of the foregoing classes.390. A combination according to claim 377 wherein the signalinginhibitor is selected from antibodies targeting EGFR; EGFR tyrosinekinase inhibitors; an antibody that targets the VEGF/VEGF receptorsystem; PDGFR inhibitors; Raf inhibitors; and PKB pathway inhibitors.391. A combination according to claim 377 wherein the ancillary agent isselected from a monoclonal antibody, an alkylating agent, an anticanceragent, a further CDK inhibitor and a hormone, hormone agonist, hormoneantagonist or hormone modulating agent.
 392. A combination according toclaim 391 wherein the anticancer agent is selected from COX-2inhibitors, HDAC inhibitors, DNA methyltransferase (methylase)inhibitors and proteasome inhibitors.
 393. A combination according toclaim 392 wherein the anticancer agent is a proteasome inhibitor isbortezimib.
 394. A combination according to claim 377 wherein the two ormore further anti-cancer agents are independently selected fromhormones, hormone agonists, hormone antagonists and hormone modulatingagents; monoclonal antibodies; camptothecin compounds; antimetabolites;vinca alkaloids; taxanes; platinum compounds; DNA binders and Topo IIinhibitors; alkylating agents; signaling inhibitors; CDK inhibitors;cylcooxygenase-2 (COX-2) inhibitors; histone deacetylase (HDAC)inhibitors; DNA methylase inhibitors; and proteasome inhibitors.
 395. Acombination according to claim 377 wherein the two or more furtheranti-cancer agents are selected from alemtuzumab, chlorambucil,cyclophosphamide, vincristine, prediniso lone, fludarabine, mitoxantroneand rituximab.
 396. A combination according to claim 377 wherein the twoor more further anti-cancer agents are selected from cyclophosphamide,doxorubicin/hydroxydaunorubicin, vincristine/Onco-TCS (V/O),prednisolone, methotrexate, cytarabine, bleomycin, etoposide,rituximab/rituxamab, fludarabine, cisplatin, and ifosphamide
 397. Acombination according to claim 377 wherein the combinations furtherinclude an additional agent selected from erythropoietin (EPO),granulocyte macrophage-colony stimulating factor (GM-CSF),granulocyte-colony stimulating factor (G-CSF), zoledronate, pamidronate,ibandronate, dexamethazone, prednisone, predniso lone, leucovorin,folinic acid and megestrol acetate.
 398. A combination according toclaim 377 wherein the ancillary compound is selected from: (a)epothilones; (b) aurora inhibitors; (c) Hsp90 inhibitors; (d) tyrosinekinase inhibitors; (e) EGF antibodies; (f) decitabine and azacytidineDNA methyl transferase inhibitors; (g) cytokines and cytokine activatingagents; (h) retinoids and rexinoids; (i) selective immunoresponsemodulators; (j) checkpoint targeting agents; (k) DNA repair inhibitors;(l) inhibitors of G-protein coupled receptor inhibitors; and (m) acombination of two or more of the foregoing classes (a) to (1).